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Ruthenium(II) 1,4,7-trithiacyclononane complexes of curcumin and bisdemethoxycurcumin: Synthesis, characterization, and biological activity.
ISSN 2412-0324 (English ed. Online)
AGRICULTURAL BIOLOGY,
2022, V. 57, Iss. 6, pp. 1166-1177
[SEL’SKOKHOZYAISTVENNAYA BIOLOGIYA] ISSN 0131-6397 (Russian ed. Print)
ISSN 2313-4836 (Russian ed. Online)
UDC 636.085.19:573.6.086.83:577.21
doi: 10.15389/agrobiology.2022.6.1166eng
doi: 10.15389/agrobiology.2022.6.1166rus
A NEW PRODUCER OF A RECOMBINANT AFLATOXIN-DEGRADING ENZYME OBTAINED VIA HETEROLOGOUS EXPRESSION
IN Pichia pastoris
I.G. SINELNIKOV1, 2, I.N. ZOROV2, Yu.A. DENISENKO1, 2, O.D. MIKITYUK1,
A.P. SINITSYN1, 2, L.A. SHCHERBAKOVA1
1All-Russian Research Institute of Phytopathology, 5, ul. Institute, Bolshie Vyazemy, Odintsovsky District, Moscow
Province, 143050 Russia, e-mail mod-39@list.ru, larisavniif@yahoo.com ( corresponding author);
2Federal Research Center Fundamentals of Biotechnology RAS, 33/2, Leninskii prospect, Moscow, 119071 Russia,
e-mailsinelnikov.i@list.ru, inzorov@mail.ru, denisenkoyura@mail.ru, apsinitsyn@gmail.com
ORCID:
Sinelnikov I.G. orcid.org/0000-0001-6359-1125
Mikityuk O.D. orcid.org/0000-0003-2022-7256
Zorov I.N. orcid.org/0000-0001-6888-172X
Sinitsyn A.P. orcid.org/0000-0001-6429-8254
Denisenko Yu.A. orcid.org/0000-0003-2363-0374
Shcherbakova L.A. orcid.org/0000-0003-0254-379X
The authors declare no conflict of interests
Acknowledgements:
Supported financially by the Russian Scientific Foundation (project No. 22-16-00153)
Received September 20, 2022
Abstract
Contamination of food and feed with mycotoxins causes significant economic losses in the
food and feed industry and poses a serious threat to the human health and animal life because of
mutagenic, carcinogenic and other disruptive properties of these secondary metabolites of fungi. Enzymatic degradation of mycotoxins represents an efficient and environmentally safe alternative to the
chemical decontamination of agricultural and food products. In this study, a synthetic adtz gene encoding ADTZ, an aflatoxin-degrading oxidase from Armillaria tabescens, was integrated into the genome of a Pichia pastoris GS115 strain under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter. To amplify the adtz gene, oligonucleotide sequences were constructed with specific
restriction sites HindIII and NotI added to the 5' end. The adtz gene-containing pPIG-ADTZ plasmid
obtained with the use of the pPIG-1 vector was linearized by digestion with restriction endonuclease
ApaI, followed by transforming the cells of P. pastoris recipient strain GS115 by electroporation. The
transformed yeast cell were selected on YPD medium with an antibiotic. PCR amplification, restriction
analysis and Sanger sequencing confirmed insertion of the target gene. As a result, 54 transformed
clones containing the target gene were obtained, and the most productive clone secreting the recombinant ADTZ-14 (2.1 mg/ml of the total extracellular protein) was selected. Recombinant ADTZ
represented a monomeric protein (78±3 kDa) possessing a high affinity to aflatoxin B1 (AFB1). Saving
the functional properties of the recombinant protein was shown using experiments on assessment of its
ability to degrade AFB1 during short-time and prolonged incubation. The obtained protein was able to
degrade AFB1 by 14 % after a 2-h incubation at 40 С; after 72 and 120 h of incubation at 30 С, the
content of AFB1 in ADTZ-14 culture liquid (CL) reduced by 50 and 80 %, respectively, compared to
content in CL of non-transformed control GS115. These data suggest a quite high biotechnological
potential of a new recombinant ADTZ preparation in relation to the decontamination of agricultural
products contaminated with AFB1. Thus, the earlier developed expression system intended to increase
the copy number of heterologous genes in Pichia pastoris was first used to obtain a recombinant
protein able to degrade AFB 1. Using this approach, we transformed yeast cells with the pPIG-ADTZ
plasmid and obtained 154 recombinant clones of P. pastoris, 77 % of which contained the target
sequence of the adtz gene. Productivity of the best transformant (clone ADTZ-14) was 2.1 mg of
protein per 1 ml of culture liquid, and about half of the pool of the extracellular proteins fell to the
share of recombinant ADTZ able to degrade 80 % of AFB 1 incubated in cell-free culture broth at
30 С and pH 7.0.
Keywords: aflatoxin B1, mycotoxins, enzymatic degradation, ADTZ from Armillaria tabescens, synthetic adtz gene, recombinant proteins, heterologous expression, Pichia pastoris
Aflatoxins, a group of structurally similar secondary metabolites of the
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fungal genus Aspergillius widely distributed in nature, are known as dangerous mycotoxins that contaminate feed and other agricultural products [1-4]. Currently,
more than 20 aflatoxins (AF), their derivatives and closely related compounds
have been identified [5]. Contamination of feeds for livestock and poultry with Band G-type AF raises the most serious concern [6, 7]. These mycotoxins are derivatives of difuranocoumarin which have a bifuran group linked to the coumarin
core and a cyclopentane (in B-type AF) or lactone ring (in G-type AF) [8, 9].
Due to the toxicity, carcinogenicity and mutagenicity of these compounds, and
their resistance to heat treatments [10, 11], feed and other crop products contaminated with AF above the concentrations allowed by hygienic regulations are not
suitable for direct use or further processing into food products. Globally, contamination with these mycotoxins, especially AFB1 which surpasses all other AFs in
hepatotoxicity and danger to warm-blooded animals [6, 7], causes serious economic damage to both agriculture and the food industry, and also create risks for
human health [1, 3].
For decontamination, physical, chemical, and microbiological methods
are used, which, however, have a number of well-known limitations [1, 12]. Therefore, there is a constant search for other effective, environmentally friendly means
and methods of AF degradation and detoxification that do not affect the quality
of agricultural products. From this point of view, an approach based on the ability
of a number of fungi [13-15] and bacteria [16-19] to synthesize enzymes that
transform AF to non-toxic or less toxic compounds seems to be very promising
[20, 22]. The use of cell-free preparations containing such enzymes makes it possible to avoid problems that may arise when using the producers themselves (for
example, deterioration of the organoleptic properties of processed products, a decrease in their nutritional value). In addition, enzyme preparations are technologically more convenient for feed processing [23] and, unlike those for the food
industry, do not require expensive multi-stage purification of the target product.
It is known that some xylotrophic basidiomycetes of the genera Pleurotus
[24, 25], Phanerochaete, and Armillaria [26-28] can be sources of enzymes for AF
degrading and detoxifying. An enzyme with oxidase activity [28] called by the
authors aflatoxin-detoxifizyme (ADTZ) was isolated from the mycelium of Armillaria tabescens using hydrophobic and metal chelate chromatography. It turned
out that ADTZ can catalyze the opening and subsequent hydrolysis of the difuran
ring [29], a structure associated with B-type AF toxicity. Further studies have
shown that ADTZ is a 76 kDa monomeric protein with high affinity for AFB1
[29]. Upon contact with ADTZ, the toxicity and mutagenicity of AFB1 were significantly reduced [28, 30].
These data indicate the prospects for the development of detoxifying drugs
containing ADTZ. However, their creation is primarily hampered by the lack of
available technology for obtaining intracellular ADTZ from A. tabescens mycelium
and, in part, by the fact that deep cultures of A. tabescens requires liquid media of
complex composition, including very specific and expensive components [31], or
a multi-stage fermentation procedure [28]. These obstacles could be overcome by
using a heterologous expression system and creating an accessible producer of the
recombinant ADTZ protein. However, there is still no suitable system for obtaining extracellular heterologous ADTZ in an amount sufficient for its use in decontamination of crop products. Nevertheless, a number of modern works [32, 33]
report on the successful use of Pichia pastoris yeast cells as recipients for heterologous expression.
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Previously, we adapted the expression system in P. pastoris by modifying
the integration vector to increase the copy number of heterologous genes in the
yeast chromosome (the integration vector and its preparation are patented) [34].
In the present study, this approach was used for the first time to create a new
producer of the aflatoxin-degrading enzyme.
Our goal was to optimize and use this system for the heterologous expression of ADTZ in Pichia pastoris GS115 and to evaluate the ability of a cell-free
culture liquid (CL) preparation of the resulting P. pastoris ADTZ-14 strain containing the extracellular recombinant ADTZ enzyme to degrade AFB1.
Materials and methods. For the expression of the adtz gene encoding aflatoxin-detoxifizim, the yeast strain Pichia pastoris GS115 (syn. Komagataella phaffii)
(Thermo Fisher Scientific, USA) was used. Yeast cells were cultured for 3 days at
30 С in liquid YPD medium (glucose 20.0 g/l; yeast extract 10.0 g/l; meat peptone 20.0 g/l). For plasmid DNA, Escherichia coli XL1-Blue (Agilent, USA) was
grown at 37 С in Luria-Bertrani medium (tryptone 10 g/l; yeast extract 5 g/l;
NaCl 5 g/l; pH 7.2-7.5). The pPIG-1 plasmid was used to express ADTZ [34]. The
adtz gene encoding the aflatoxin degradation enzyme in A. tabescens (GenBank
AY941095.1) was synthesized at ZAO Evrogen (Russia) according to the codon
compositions in P. pastoris.
PCR mix for the adtz gene amplification (50 µl) contained 1½ buffer with
3 mM MgCl2 and 5 U Taq polymerase (NEB, UK), 0.2 μM ADTZ-fwd (5´-gaagcttctATGGCTACTACAACTG-3´) and ADTZ-rev (5´-cgcggccgcTTACAATCTTCTCTC-3´) oligonucleotides, and 0.1 ng DNA as a matrix. The reaction was
carried out under the following conditions: 95 С for 15 s, 62 С for 15 s, and
72 С for 120 s (25 cycles) (a T-100 amplifier, Bio-RAD, USA). The PCR products were evaluated electrophoretically (a 1% agarose gel, a Sub-Cell GT Cell,
Bio-RAD, USA).
The amplification product, vector pPIG-1, was digested with HindIII and
NotI restriction endonucleases according to the manufacturer’s recommendations
(NEB, UK).
The processed fragments were ligated with T4 DNA ligase (ZAO Evrogen, Russia), and Escherichia coli XL1-blue cells (Agilent, USA) were transformed with the 2 μl mixture by the heat shock method. Transformants were
selected on Luria-Bertrani agar medium containing ampicillin (100 μg/ml). The
pPIG-ADTZ plasmid was isolated from ampicillin-resistant transformants using
the Plasmid Mini-prep kit (ZAO Evrogen, Russia). The presence of the target
gene insert in the pPIG-ADTZ plasmid was confirmed by PCR amplification,
restriction analysis as described above, and Sanger sequencing. Sequencing was
performed in both directions from primers used for the gene amplification. Gene
sequencing and synthesis of primers used for amplification were performed at
OOO Sintol (Russia).
The pPIG-ADTZ plasmid was linearized by digestion with restriction endonuclease ApaI (NEB, UK) according to the manufacturer's protocol and transferred into P. pastoris GS115 by electroporation [35]. Transformants were selected
on a YPD agar medium supplemented with 200 μg/ml antibiotic zeocin (Thermo
Fisher Scientific, USA). DNA was isolated from antibiotic-resistant colonies [36]
and the ADTZ insert was checked by PCR.
The recombinant ADTZ protein was produced by culturing the P. pastoris
ADTZ-14 producer strain in 24-well plates (3 ml YNB liquid medium, 30 С,
aeration 200 rpm, 3 days). Every 24 hours, 40% glucose solution in 20 mM
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potassium phosphate buffer (pH 6.0) was added to the wells to a final concentration of 2%.
The recombinant ADTZ in cell-free CL was detected by polyacrylamide
gel electrophoresis with sodium dodecyl sulfate (DNS-PAGE, Mini-PROTEAN®
Tetra, Bio-RAD, USA). Total protein concentration was measured by the Lowry
method [37].
The degradation kinetics of AFB1 and AFG1 was studied in experiments
with short-term incubation of P. pastoris ADTZ-14 cell-free CL. Commercial
preparations of AFB1 and AFG1 (VNIIVSGE, Russia) were dissolved in 20 mM
Na-phosphate buffer (pH 6.7) to a final concentration of 2.5 μg/ml each. The
concentration was controlled using the molar extinction coefficients = 21800
and = 17700 (at = 362 nm) for AFB1 and AFG1, respectively. The CL of the
P. pastoris ADTZ-14 transformant was incubated with toxins in the wells of a
thermostated autosampler plate (30 or 40 С). A 5 µl aliquots were taken from the
reaction mixture every 30 min for 2.5 h and the AF content was determined by
reverse-phase chromatography on a thermostated (30 С) Kromasil Ethernity 5C18 column (4.6½250 mm) (Akzo Nobel, Sweden) equipped with an appropriate
guard column. An Agilent 1200 chromatographic system (Agilent Technologies,
USA) with diode array detection was used. Chromatographic separation was performed in a water/acetonitrile gradient (from 40% to 68% acetonitrile in 20 min,
detection at 360, 235, and 225 nm, slit width 8 nm). The degree of AFB1 and AFG1
degradation was assessed by the change in the area of the corresponding chromatographic peak. The CL of the untransformed strain P. pastoris GS115 was a control.
To assess the ability of the recombinant ADTZ enzyme to degrade AFB1
during prolonged incubation, 1 ml of P. pastoris ADTZ-14 CL samples (2.1 mg
total protein/ml) after pre-sterilization by filtration (membranes with a pore size
of 0.22 μm, Millipore, USA) were added with 1.0 μg of AFB1 (Sigma-Aldrich,
USA) dissolved in a minimum volume of methanol. Samples (1 ml) of CL of nontransformed strain P. pastoris GS115 addedd with the same amount of AFB1 were
used as a control. Samples were incubated for 3 and 5 days at pH 7.0 and 30 С.
The post-incubztion AFB1 concentration was measured using high-performance
liquid chromatography (HPLC) on a thermostated (27 С) Symmetry C18 column (5 µm, 150½4.6 mm) in isocratic elution mode (mobile phase methanol:water
60:40, 10 µl sample injected, λ = 362 nm) using a Waters 1525 Breeze system with
a Waters UV 2487 detector (Waters Corp., USA) [12, 38]. Prior to HPLC analysis,
CL samples were diluted 100-fold with the mobile phase. Toxin concentrations
were measured in the linear detection range in the test and control samples, The
concentrations were calculated from the calibration curve for the AFB1 standard
(Sigma-Aldrich, USA). The percentage of degradation was determined relative to
the amount of toxin detected in the corresponding control sample.
Statistical processing of AFB1 quantification data was performed using the
STATISTICA 6.1 program (StatSoft, Inc., USA). Significance of differences at
p 0.05 was confirmed using Student’s t-test for independent variables. The table
and figures indicate the mean values (M) of two measurements for each of the
three biological repetitions with standard deviations (±SD).
Results. The gene for the aflatoxin-degrading enzyme from A. tabescens
was cloned by PCR using the developed oligonucleotides.
The size of the amplification product corresponding to the synthesized
adtz gene was 2088 bp. Sequencing of the obtained product confirmed its identity
with the sequence of A. tabescens (GenBank AY941095.1) (Fig. 1).
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Fig. 1. Sequence alignment visualization of the codon-optimized adtz gene and the natural adtz gene
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of Armillaria tabescens (GenBank AY941095.1, https://www.ncbi.nlm.nih.gov/genbank/). Optimization of the codon composition was carried out by ZAO Evrogen (Russia) using the codon frequency
table for Pichia pastoris (https://www.kazusa.or.jp).
Fig. 2. A map of the pPIG-ADTZ plasmid obtained by cloning the sequence of the synthesized aflatoxindetoxphyzyme (ADTZ) adtz (synthetic_ADTZ) gene into the pPIG-1 vector.
The resulting recombinant gene was integrated into the pPIG-1 vector
by the restriction ligation method. Restriction analysis of the resulting new plasmid, after double digestion with HindIII and NotI, resulted in 5500 and 2100 bp
products, confirming the correct integration of the target sequence into the pPIG1 vector. The resulting recombinant plasmid (Fig. 2) was called pPIG-ADTZ.
Plasmid pPIG-ADTZ, linearized with restriction endonuclease ApaI, was
electroporated into competent P. pastoris GS115 cells, and transformants were
selected on YPD medium added with zeocin. Cloning resulted in 154 clonal colonies. Genomic DNA isolated from 70 randomly selected transformed clones was
analyzed by PCR to identify the adtz gene insert. PCR analysis of the DNA of
these transformants grown on the selective media showed that at least 54 clones
contained the target adtz insert. Among them, the ADTZ-14 clone turned out to
be the most productive and was used for further work. Already after 72 h of culture,
expression of ADTZ in this clone led to the accumulation in the CL of the extracellular recombinant protein. The size of this protein according to the SDS-PAGE
analysis was 78±3 kDa (Fig. 3), while in the CL of the nontransformed recipient
P. pastoris GS115 we did not find proteins of a comparable size. In the CL of the
transformed ADTZ-14 clone, the total protein concentration was 2.1 mg/ml.
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Fid. 3. Electrophoregram of the culture liquid proteins of the strain
Pichia pastoris ADTZ-14 (1) transformed with the recombinant
pPIG-ADTZ vector containing the synthesized aflatoxin-detoxifizyme (ADTZ) adtz gene, and the recipient P. pastoris GS115 (2).
M — molecular weight marker PageRuler™ 26614 (Thermo
Fisher Scientific, USA).
Comparison of the degradation kinetics of
AFB1 and AFG1 in CL of P. pastoris ADTZ-14
showed that the recombinant enzyme is capable
of destroying both toxins; however, its efficiency
against AFB1 turned out to be significantly higher
than for AFG1. Thus, under the action of CL of
the producer of recombinant extracellular ADTZ,
already after 2 h of incubation, the concentration
of AFB1 toxin decreased by approximately 14%
compared to the initial level, while for AFG1 the decrease was only 4% (Fig. 4).
Fig. 4. Degradation kinetics of aflatoxins G1
(1) and B1 (2) in the cell-free culture fluid
of the Pichia pastoris ADTZ-14 strain transformed with the pPIG-ADTZ recombinant
vector conyaining the synthesized aflatoxindetoxphyzyme (ADTZ) adtz gene at 40 С
and pH 6.7 (n = 3, М±SD).
The obtained results were
consistent with the data of other
authors who noted the high specificity of intracellular ADTZ from
A. tabescens to AFV1 [29]. In this regard, we studied the degradation activity of
recombinant ADTZ with respect to the indicated toxin during its longer incubation
with cell-free CL of the P. pastoris ADTZ-14 strain.
Fig. 5. Chromatograms of culture liquid (CL) samples of Pichia pastoris (incubation at 30 С and pH 7.0).
A: CL of the non-transformed recipient strain GS115 (control).
B: CL GS115 + aflatoxin B1 (APB1, 1 µg/ml) after incubation (control sample). The peak
on the chromatogram corresponds to 10 ng of AFB1 in the injected sample.
C and D: CL of the P. pastoris ADTZ-14 strain transformed with the pPIG-ADTZ recom-
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binant vector containing the synthesized aflatoxin-detoxiphyzyme (ADTZ) adtz gene, + AFV1
(1 μg/ml) after 3 and 5 days of incubation, respectively.
In these experiments, it was found that after 3 days the concentration of
the toxin added to the CL decreased by almost 2 times, and after 5 days the
efficiency of its degradation reached 80% (Fig. 5, Table).
Dynamics of enzymatic degradation of aflatoxin B1 (AFB1) in the cell-free culture
liquid (CL) of the Pichia pastoris ADTZ-14 transformant straindecreting the recombinant aflatoxin-detoxphyzyme (ADTZ) (n = 6, M±SD)
Strain
0h
AFB1, g/ml
Incubation time
72 h
120 h
AFB1, g/ml degradation, % AFB1, g/ml degradation, %
ADTZ-14
0.97±0.01
0.57±0.06
41.2а
0.19±0.04
80.4b
GS115 (control)
0.98±0.01
1.01±0.01
0.0с
0.90±0.05
0.1с
N o t е. The ADTZ-14 strain was obtained by transformation of the P. рastoris GS115 recipient with the pPIGADTZ recombinant vector with the synthesized aflatoxin-detoxphyzyme (ADTZ) adtz gene. Before incubation,
AFB1 was added to the culture liquid (CL) to a concentration of 1 μg/ml; for 0 h, the concentrations detected in
the CL samples before incubation are indicated (opening from 96 to 99%).
abc Differences between percent degradation marked with different letters are statistically significant at p 0.05.
The data we presented here indicate a rather high biotechnological potential of the new producer of recombinant ADTZ and expand the so far limited
spectrum of recombinant enzymes of other xylothorophic fungi degrading AFB1
obtained using the system of heterologous expression in P. pastoris [39].
It should also be noted that the ADTZ-14 producer was characterized by
a rather high level of expression of extracellular proteins for P. pastoris. Probably,
the use of a synthetic gene with optimized codons contributed to an increase in
the productivity of yeast cells. A similar approach has been successfully used previously for the expression of bacterial -amylase in P. pastoris [40]. However,
according to recent data, the use of synthetic genes can lead to protein misfolding,
degradation, and a decrease in its activity and stability. This may be the cause of
partial degradation of secreted recombinant proteins [41], which, as noted above,
we also observed in our experiments with electrophoretic analysis of the CL of the
transformed clone ADTZ-14. Therefore, it is necessary to continue research on
increasing the efficiency of AFB1 degradation by recombinant ADTZ, additional
testing of the effect of this recombinant enzyme on other aflatoxins, as well as
experiments on the treatment of crop products contaminated with AFB1 with the
enzyme preparation. It is also possible that heterologous expression using other
eukaryotes, e.g., filamentous fungi which are used for bioprocessing of feed to
increase its nutritional value, will allow for obtaining new producers of highly
active extracellular ADTZ. Such producers could be promising for the simultaneous decontamination of plant raw material contaminated with aflatoxin and increasing the availability of their nutritional components.
Thus, this article is the first report on the production of a recombinant
protein capable of cleaving AFB1 using an expression system developed by us
earlier to increase the copy number of heterologous genes in Pichia pastoris. Yeast
cells were transformed with the pPIG-ADTZ plasmid and 154 recombinant clones
of P. pastoris were generated, 77% of which contained the target sequence of the
ADTZ synthetic aflatoxin-detoxiphyzyme adtz gene. The protein yield of the most
productive ADTZ-14 transformant was 2.1 mg/ml cell-free culture liquid, and in
this case, about half of all extracellular protein pool was recombinant ADTZ.
Incubation of AFB1 with this recombinant ADTZ led to 80% degradation of the
added toxin. The transformant strain P. pastoris ADTZ-14, secreting functional
ADTZ, can be a producer of an accessible and sufficiently active recombinant
enzyme for AFB1 degradation. Based on P. pastoris ADTZ-14, preparations for
the enzymatic degradation of AFB1 can be developed in the future. Confirmation
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of the decontamination potential of the recombinant enzyme will indicate the
feasibility of optimizing biotechnology to increase the yield of the target product
and develop its formulation.
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