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BIOTECHNOLOGY
SCREENING AND IDENTIFICATION OF PIGMENTAL YEAST PRODUCING L-PHENYLALANINE AMMONIA-LYASE AND THEIR PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERISTICS
O. O. Babich
Kemerovo Institute of Food Science and Technology, bul'v. Stroitelei 47, Kemerovo, 650056 Russia phone: +7 (3842) 68-06-83, e-mail: olich.43@mail.ru
(Received March 31, 2014; Accepted in revised form April 30, 2014)
Abstract: The results of the analysis of DNA sequences encoding L-phenylalanine ammonia-lyase (PAL) synthesis, performed to obtain universal primers complementary to conserved regions of the pal gene, are presented in the article. The fragment of pal gene was amplified in organisms under study. Nucleotide sequence of the pal gene in microorganisms exhibiting L-phenylalanine ammonia-lyase activity was determined by DNA sequencing. The results of its comparison with the corresponding sequences of known species are presented. Phenotypic characteristics and biochemical properties of selected cultures were studied. An investigation aimed to choose a superproducer strain of L-phenylalanine ammonia-lyase was conducted. It was found that L-phenylalanine ammonia-lyase synthesis was the most active in the following strains: Aureobasidium pullulans Y863, Rhodosporidium infirmominiatum Y1569, Candida glabrata Y2813, Candida maltose Y242, Debaryomyces robertsiae Y3392, Rhodosporidium diobovatum Y1565, Rhodotorula lactose Y2770, Saccharomyces cerevisiae Y1127, Tilletiopsis washingtonensis Y1650, Torulopsis apicola Y566, Tremella foliacea Y1624, Rhodotorula rubra Y1193, and Debaryomyces castellii Y968. This allows recommending them for further research aimed to obtain the enzyme preparation of L-phenylalanine ammonia-lyase.
Keywords: L-phenylalanine ammonia-lyase, enzyme, pal gene, pigmental yeast, nucleotide sequence, amino acid
sequence, phylogenetic tree
UDC 579.67 DOI 10.12737/5464
INTRODUCTION
L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) catalyzes the reaction of reverse deamination of L-phenylalanine to trans-cinnamic acid and ammonia [1]. It is the key enzyme of phenylpropanoid metabolism in plant and fungi, where it is involved in biosynthesis of secondary metabolites (flavonoids, furanocoumarines, and cell wall components), existing in multiple isoforms [2, 3].
PAL plays an important role in catabolic processes of microorganisms, providing for utilization of L-phenylalanine as a sole source of carbon and nitrogen [4]. Among the microorganisms, the highest PAL activity is exhibited by yeasts, especially the red basidiomycetes of the Rhodotorula family [5]. Sporobolomyces roseus and Sporidiobolus pararoseus are also PAL-producing yeasts [6].
Therapeutic potential of PAL with respect to neoplasms was evaluated due to its selective activity to phenylalanine and amino acids that are consumed by mammalian cells from external sources. PAL was shown to inhibit neoplasm growth in vitro [7].
The enzyme is also of interest as a therapeutic agent for phenylketonuria treatment and may be used for both direct therapy of phenylketonuria and production of food products free of phenylalanine [8]. Besides the medical applications, the enzyme may be used in
biotechnology for L-phenylalanine production from trans-cinnamic acid [9].
Considerable contribution to the development and assimilation of the technology of specialized food products was made by G.B. Gavrilov, N.B. Gavrilova, V.I. Ganina, N.I. Dunchenko, I.A. Evdokimov, V.I. Kruglik, K.S. Ladodo, L.A. Ostroumov,
A.N. Petrov, V.O. Popov, G.Yu. Sazhinov,
V.A. Tutel'yan, V.D. Kharitonov, I.S. Khamagaeva, and A.G. Khramtsov, and to the technology of the enzyme preparation of PAL, by V.I. Mushtaev,
M. Jason Мас Donald, H. Orum, and O.F. Rasmussen.
The development of new and improvement of existing technologies of the PAL preparation production requires new, more intensive sources of its superexpression, which is impossible without studies on the specific features of its genetics in known producers. Only 26 sequences of genomes of microorganisms exhibiting PAL activity were found in the databases of genetic sequences (EMBL and GenBank). Therefore, the search for microorganisms exhibiting L-phenylalanine ammonia-lyase activity based on sequence analysis of their genomes is urgent.
The aim of the work was to screen and identify pigmented yeasts producing L-phenylalanine ammonia-lyase and describe their physiological and biochemical characteristics.
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OBJECTS AND METHODS OF RESEARCH
Agarose (Chemapol, Czech Republic); low gelling-temperature agarose (Ultra Pure, BRL, United States); P-mercaptoethanol, PEG-1500, PEG-6000 (Loba
Feichemie, Austria); imidazole (Diaem, Russia); dATP, dCTP, dGTP, dTTP (Bioline, Germany); bactoagar, yeast extract (Difco, United States); bactotriptone (Ferak, Germany); D-glucose, urea, bromine (99.8%, for synthesis) (Merck, Germany); bromphenol blue, ammonium persulfate, N,N-methylene-bis-acrylamide (Reanal, Hungary); acetyl phosphate, ethidium bromide, DTT, IPTG, Triton X-100, EDTA, MgCl2, BSA, DMSO, Tris, (NH4)2SO4, CaCl2 (Sigma, United States); L-phenylalanine ammonia-lyase, acrylamide, N,N-methylene-bis-acrylamide, ammonium persulfate (Sigma, Germany); RNase A, CH3COOK, CeH5OH, CHCl3, NaCl (Reakhim, Russia); MilliQ deionized water (Millipore, France); orthophosphoric acid (85.1%, imported) (Lenreaktiv, Russia); tris(hydroxymet-hyl)aminomethane, PIPES, SDS, X-Gal, tetracycline hydrochloride, chloramphenicol (Serva, Germany); L-phenylalanine (Acros Organics, Belgium); transcinnamic acid (Briture Co. Ltd., China); boric acid (99.8%, extra pure), sodium tungstate dihydrate (99.1%, extra pure) (AppliChem, United States); (3-amino-propyl)triethoxysilane (98.0%, for synthesis), acrylic acid (99.8%, for synthesis), sodium caseinate (92.0%, extra pure), potassium monophosphate dihydrate (98.5%, extra pure), sodium carbonate (99.9%, for synthesis), sodium hydroxide (9.1%, pure for analysis), sodium chloride (99.8%, extra pure), hydrochloric acid (36.0%, extra pure) (Khimlaborpribor, Russia); sodium phosphate dibasic dodecahydrate (98.2%, extra pure), L-tyrosine (99.9%, extra pure), trichloroacetic acid (99.0%, for synthesis), acetic acid (98.5%, extra pure), phenolphthalein (98.5%, extra pure).
Sequences of the pal gene of ascomycetes and basidiomycetes presented in the NCBI international database are reported in Table 1.
Table 1. Sequences of microorganism strains possessing L-phenylalanine ammonia-lyase activity
Strain Strain number Species name
1 83976309 Rhodotorula graminis
2 3293 Rhodosporidium toruloides
3 3284 Rhodotorula mucilaginosa
4 317034460 Aspergillus niger
5 115437191 Aspergillus terreus
6 497418 Arabidopsis thaliana
7 242780352 Talaromyces stipitatus
8 212533750 Penicillium marneffei
9 169610841 Phaeosphaeria nodorum
10 317157281 Aspergillus oryzae
11 238493630 Aspergillus_flavus
12 121698870 Aspergillus clavatus
13 119480760 Neosartorya_fischeri
14 71001127 Aspergillusfumigatus
15 389639669 Magnaporthe oryzae
16 116206211 Chaetomium globosum
17 164422921 Neurospora crassa
18 15824530 Ustilago maydis
Table 1. Ending. Sequences of microorganism strains possessing L-phenylalanine ammonia-lyase activity
Strain Strain number Species name
19 331236172 Puccinia graminis
20 507833891 Letharia vulpina
21 4127288 Amanita muscaria
22 299751359 Coprinopsis cinerea okayama
23 170097945 Laccaria bicolor
24 409924409 Tricholoma matsutake
25 482667462 Pleurotus eryngii
26 1666264 Agaricus bisporus
Strains of the microorganisms for study of their L-phenylalanine ammonia-lyase activity obtained form the All-Russian Collection of Industrial Microorganisms, GosNIIGenetika, are presented in Table 2.
Table 2. Strains of the microorganisms for investigation of the PAL activity
No. Name Microorganism number in the Collection of Microorganisms of GosNIIGenetika
1 Aureobasidium pullulans Y863
2 Bullera alba Y1581
3 Bullera piricola Y1577
4 Candida glabrata Y2813
5 Candida maltosa Y242
6 Cryptococcus laurentii Y227
7 Cryptococcus macerans Y2763
8 Cystofilobasidium capitatum Y1573
9 Cystofilobasidium capitatum Y1852
10 Debaryomyces castellii Y968
11 Debaryomyces robertsiae Y3392
12 Dioszegia hungarica Y3208
13 Dioszegia sp. Y3320
14 Geotrichum klebahnii Y3053
15 Phaffia rhodozyma Y1666
16 Phaffia rhodozyma Y1668
17 Rhodosporidium capitatum Y1567
18 Rhodosporidium diobovatum Y1565
19 Rhodosporidium infirmo-miniatum Y1569
20 Rhodotorula aurantiaca Y985
21 Rhodotorula glutinis Y77
22 Rhodotorula lactosa Y2770
23 Rhodotorula minuta Y2777
24 Rhodotorula rubra Y1193
25 Saccharomyces cerevisiae Y1127
26 Saccharomyces kluyveri Y2559
27 Sporobolomyces holsaticus Y991
28 Sporobolomyces roseus Y987
29 Tilletiopsis washingtonensis Y1650
30 Torulopsis apicola Y566
31 Tremella foliacea Y1624
32 Tremella mesenterica Y1625
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BLAST2 software was used to search for homologous sequences; Generunner and Chromas, for analysis of nucleotide and amino acid sequences; Clustal Omega, for multiple nucleotide sequence alignment; and BioEdit 7.0.0, for editing and alignment, as well as translation into amino acid sequence. Analysis of an optimal model for amino acid substitutions was performed on a ProtTest 3 on-line server according to the Akaike information criterion (AIC) (the sequence of the pal gene of Arabidopsis thaliana was used as an out-group). The phylogenetic tree was built using a distance method in a Phylip 3.69 software package, and using the Bayesian method, in a MrBayes 3.2 software; the trees were visualized in a TreeGraph 2, and the logo diagram was built in a WebLogo software.
Sanger sequencing was performed on an ABI3730xl (Applied Biosystems, United States) automated sequencer according to the manufacturer’s protocol using the BigDye® Terminator v3.1 Cycle Sequencing kit.
Oligonucleotides were obtained on an ABI3900 (Applied Biosystems) synthesizer. The results of the experiments were processed using the methods of mathematical statistics.
The primers RalF and RalR were synthesized by JSC Sintol. Primer operational parameters are presented in Table 3.
Table 3. Operational parameters of RalF-RalR primers
Primers
Forward primer RalF CTCACCAACTTCCTCAA CCACGGCA
Parameters
Length 25 bp
Molecular weight 7475.9
CG content 56%
Melting temperature 69°С
Annealing temperature 64°С
Reverse primer RalR ATGCCCTCGTCGTCCTT GACCTTGA
Parameters
Length 25 bp
Molecular weight 7559.9
CG content 56%
Melting temperature 69°С
Annealing temperature 64°С
PCR amplification was performed on a SMART CYCLER (Cepheid, United States) thermocycler in 20-50 ^L solution prepared on the basis of a 10-fold buffer for Taq polymerase, which contained 200 ^mol of each of deoxyribonucleotides, 0.5 ^mol primers, 2 ^mol MgSO4, 10 ng template, 2 units of Taq DNA polymerase, and 0.1 unit of Pfu DNA polymerase. Oligonucleotide annealing temperature was calculated using an empirical formula: Tm = 67.5 + 34[%GC] -395/n, where %GC = (G + C)/n, and n is the number of nucleotides. PCR products were analyzed by electrophoresis in 1-% agarose gel.
Table 4 presents the amplification parameters for RalF-RalR primers.
Table 4. Amplification parameters for RalF-RalR primers
Step Temperature, °C Time
Initial heating 90-95 1 min
Denaturation 95 30 s 30 cycles
Primer annealing 64 30 s
Elongation 72 1 min
Elongation 72 1 min
DNA electrophoresis in agarose gel. Samples of DNA were separated by electrophoresis in Tris-acetate buffer (0.04 M Tris-acetate; 0.002 M EDTA) in a 0.70.8% agarose gel (Bio-Rad, United States) containing 0.5 ^g/mL ethidium bromide under the voltage of 2-5 V/cm. GeneRuler™ 1 kb DNA Ladder (Fermentas, Lithuania) were used as standard molecular weight markers. DNA bands were detected upon gel irradiation with UV light using the Gel Doc XR Plus (Bio-Rad) system.
Isolation of DNA fragments from agarose gel.
Samples of DNA were separated by electrophoresis in Tris-acetate buffer in a 0.7-0.8% agarose gel (BioRad) containing 0.3 ^g/mL ethidium bromide and analyzed by fluorescence under ultraviolet light at 254 nm. Gel pieces containing fragments of interest were cut out and transferred into microcentrifuge tubes, then DNA fragments were eluted from the gel using the “Isolation of DNA from agarose gels” kit (Boeringer Mannheim, Germany). Sodium perchlorate was added to the tubes in the amount of 400 ^L per 100 mg weight of the cut out gel. The mixture was heated to 65°C, then agarose was dissolved in salt buffer. Glass milk microbeads were introduced into the suspension at the amount of 20 ^L per 100 mg of gel weight. In the salt solution, DNA contained in the gel adsorbed on the surface of the microbeads. They were washed (consecutive precipitation-resuspension) with the same salt solution once and with 70% ethanol, two times. DNA was desorbed from the beads by resuspension in TE buffer (10 mM Tris-HCl buffer, pH 7.4, 1 mM EDTA) in the amount of 50 ^L per 100 mg gel weight.
L-phenylalanine ammonia-lyase activity was determined according to a protocol by Sigma with little modifications in the preparation of the reaction mixture. All solutions were prepared in deionized MilliQ water.
Composition of standard incubation mixture (1 cm3): 0.2 mol Tris-HCl, pH 8.5, 0.5 cm3; 0.05 mol L-phenyl-alanine, 0.04 cm3; deionized water, 0.42 cm3.
After mixing and pre-incubation (at least 5 min at 30 ± 0.1°C), the reaction was initiated by the addition of 0.04 cm3 diluted enzyme (0.025-0.125 U/cm3 PAL).
In the control sample, 0.04 cm3 water was added instead of the enzyme.
The reaction course was registered continuously at 270 nm in a Shimadzu UV-1800 (Shimadzu, Japan) spectrophotometer equipped with a thermocontrolled chamber in quartz cuvettes with a 1-cm optical path. Data collection and analysis was performed by a UV-probe v (Shimadzu) software. PAL activity was calculated according to the formula using the value of millimolar extinction coefficient of trans-cinnamic acid (Sigma’s protocol):
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. . . fit і \ (ДО^270^ ^roae
Actmty \y/ctr,i)" p 19 73 x ,, ■
where Vreac.mix. is the reaction mixture volume, mL; f coefficient of dilution of the initial PAL preparation; 19.73, millimolar coefficient of extinction of transcinnamic acid at 270 nm; Vsample, sample volume, cm3; minexp, mincontrol, duration of enzymatic activity measurement in experiment and control samples, respectively.
The amount of PAL that catalyzed the transformation of 1 ^mol L-phenylalanine into trans-cinnamic acid and NH3 within 1 min at pH 8.5 at 30 ± 1°C was considered an activity unit.
Study of specific features of phenotype and biochemical properties of the selected pigmented yeast strains. Shape and size of cells of the microorganisms were described and determined in cultures of different age on dense and liquid nutritive media. The first imaging and cell size measurement was performed in two-three-day cultures grown at 25-28°C. Then, the cultures were left at room temperature (17-18°C) and described again in four weeks.
To determine cell size, length and width were measured with a micrometer in at least 20 cells, and the extreme values were indicated.
RESULTS AND DISCUSSION
Today, to determine the species reference of a microorganism and the presence of a certain gene in it, the method of 16S rRNA gene sequence comparison may be used, since the gene carries both the conserved and variable regions of the nucleotide sequence. The data on nucleotide sequences of various
microorganisms are contained in the international databases GenBank and EMBL-EBI.
Therefore, comparative analysis of twenty six nucleotide sequences of the pal gene contained in the GenBank database of genetic sequences were compared, including Rhodotorula graminis 83976309, Rhodosporidium toruloides 3293, Rhodotorula mucilaginosa 3284, CBS 513.88 Aspergillus niger 317034460, NIH2624 АspergШus terreus 115437191, Arabidopsis thaliana 497418, ATCC_10500
Talaromyces stipitatus 242780352, ATCC 18224 Penicillium marneffei 212533750, SN15 Phaeosphaeria nodorum 169610841, RIB40 Aspergillus oryzae 317157281, NRRL3357 Aspergillus flavus 238493630, NRRL 1 Aspergillus clavatus 121698870, NRRL 181 Neosartorya fischeri 119480760, Af293 Aspergillus fumigatus 71001127, 70-15 Magnaporthe oryzae
389639669, CBS_148.51 Chaetomium globosum
116206211, OR74A Neurospora crassa 164422921, Ustilago maydis 15824530, f. sp. tritici CRL 75-36-7003 Puccinia graminis 331236172, Letharia vulpina 507833891, Amanita muscaria 4127288, 7.130
Coprinopsis cinerea okayama 299751359, S238N-H82 Laccaria bicolor 170097945, NBRC 30605 Tricholoma matsutake 409924409, P810 Pleurotus eryngii
48266746, and Agaricus bisporus 1666264, deposited in the GenBank of the National Center for Biotechnology Information (NCBI).
Phylogenetic relationships between the microorganisms established on the basis of comparative analysis of the nucleotide sequences may be presented as a dendrogram (phylogenetic tree), an arbitrary graphical representation reflecting the affinity between the genetic macromolecules, biological species, or higher rank taxa (Fig. 1).
Fig. 1. Dendrogram of nucleotide sequences of the pal gene built with the NJ method using the MEGA 4.0.2 software. Figures indicate the statistical reliability of the branching order determined with bootstrap analysis.
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Figure 1 presents the molecular phylogenetic tree obtained using the neighbor-joining method. The genetic distances have been calculated according to Kimura-2 method.
The performed phylogenetic analysis demonstrates rather high similarity between the pal gene sequences. In the phylogenetic tree, all pal gene sequences under study may be grouped into two big clusters, I and II.
Cluster I comprises the smaller clusters 1, with bootstrap support of 85%, and 2, with bootstrap support of 69%; pal gene sequences of Agaricus bisporus and Pleurotus eryngii correspond to clade 1b with bootstrap support of 36%. In clade 1a, Amanita muscaria forms an individual branch, and Coprinopsis cinerea okayama, Laccaria bicolor, and Tricholoma matsutake form a small clade with support of 40%, and Laccaria bicolor forms a separate consortium with Tricholoma matsutake with the support of 83%.
Sequences of pal gene in the representatives of the Rhodotorula and Rhodosporidium genera, Rhodotorula graminis, Rhodosporidium toruloides, and Rhodotorula mucilaginosa, form a separate clade 1b in cluster 1 with the highest bootstrap support of 100%. They are neighbored by Ustilago maydis, which forms an individual branch.
Cluster II may be divided into a small cluster 3 and a large cluster 4.
Cluster 3 comprises the sequences of the represent-tatives of the Arabidopsis thaliana and Puccinia graminis species with bootstrap support of 82%.
Representatives of the Aspergillus genus—
A. oryzae, A. flavus, A. clavatus, Neosartorya fischeri (synonym with A. fischeri), and A. fumigatus—form a monophyletic clade 4b2 1 with a 100-% bootstrap support. They are neighbored by another clade with a 100-% support, which includes pal sequences of Talaromyces stipitatus and Penicillium marneffei.
Clade 4 b1 also comprises the representatives of A. niger and А. terreus, which form a monophyletic consortium with support of 99%, and Chaetomium globosum and Neurospora crassa, a consortium with bootstrap support of 100%.
Sequences of the pal gene from Magnaporthe oryzae and Phaeosphaeria nodorum form a consortium with 77-% support. The neighboring Letharia vulpine, Letharia vulpine, Magnaporthe oryzae, and Phaeosphaeria form a separate branch comprising the 4a clade with support of 33%. Clades 4a and 4b together make up cluster 4 with bootstrap support of 76%.
Multiple alignment of selected nucleotide sequences for each of the gene clusters encoding L-phenylalanine ammonia-lyase deposited in GenBank and EMBL-EBI were performed with the Clustal X V 1.75 software.
The results of the study demonstrated that the pal sequences in the analyzed microorganisms are poorly conserved; therefore, the area of search for universal primers was narrowed. For this purpose, pal gene sequences in basidiomycete yeasts Rhodosporidium toruloides and Rhodotorula glutinis were analyzed, because these very organisms possessed the highest homology with the pigmented yeast strains.
Based on the obtained conserved fragments, as well as the theoretical rules of selection, the following
universal primers were selected:
- forward primer PAL1F (5'- CGC GGY CAY TCK GCK GT -3')
- and reverse primer PAL1R (5'-CAT YTC TGC CGG YTG AAC RTG -3').
Melting temperature and amplification parameters for the designed primers were calculated using the Olig 4.0 software. The results of the studies are presented in Table 3.
Analysis of the literature data demonstrated that L-phenylalanine ammonia-lyase was detected in a number of microorganisms, including the pigmented yeasts. Colorless yeasts do not contain the enzyme. In this connection, the search for culture with PAL activity was performed among the pigmented yeast from the microorganism collection (GosNIIGenetika): Aureobasi-dium pullulans Y863, Bullera Y1581, Y1577, Candida Y2813, Y242, Cystofilobasidium Y1573, Debaryo-myces Y968, Y3392, Phaffia 1666, 1668, Rhodospo-ridium Y1567, Y1565, Y1569, Rhodotorula Y985, Y2770, Y2777, Y1193, Saccharomyces Y1127,
Y2559, Sporobolomyces Y987, Tilletiopsis Y1650, Torulopsis Y566, and Tremella Y1624, Y1625.
As a result of amplification reaction, a PCR product of expected length was obtained for each of the samples. However, we failed to sequence it, since the reaction yielded additional non-specific products of various length, including the one very close to the target fragment. We decided to substitute one of the primers with another one and decrease the length of expected fragment. At the second stage the PAL2F primer (CAT YTC TGC CGG YTG AAC RTG) was used. Therefore, the pair of primers RAL2F-RAL1R flanks the pal gene fragment located between nucleotides 292 and 1319 of the pal gene in NCBI database; the size of the amplified fragment was 1027 bp.
The results of amplification were controlled with electrophoresis in 1.5% agarose gel under voltage of 5 V/cm in an SE-1 horizontal electrophoresis chamber equipped with an Elf-4 power supply (Khelikon, Russia). Artificially synthesized pal gene of Rhodosporidium toruloides was used as a positive control.
After enzymatic purification with a mixture of exonuclease I and alkaline phosphatase, the fragments were sequenced with the PAL2R primer on an ABI 3130xl (Applied Biosystems) analyzer according to standard techniques.
To prove that the obtained amplificates are indeed the pal gene fragments, direct sequencing of PCR fragments was performed using an ABI3730xl (Applied Biosystems) automated sequencer and BigDye® Terminator v3.1 Cycle Sequencing Kit for the following strains: Aureobasidium pullulans Y863, Bullera alba Y1581, Bullera piricola Y1577, Candida glabrata Y2813, Candida maltosa Y242, Cryptococcus laurentii Y227, Cryptococcus macerans Y2763, Cystofilobasidium capitatum Y1573, Cystofilobasidium capitatum Y1852, Debaryomyces castellii Y968, Debaryomyces robertsiae Y3392, Dioszegia hungarica Y3208, Dioszegia sp. Y3320, Geotrichum klebahnii Y3053, Phaffia rhodozyma Y1666, Phaffia rhodozyma Y1668, Rhodosporidium capitatum Y1567, Rhodosporidium diobovatum Y1565,
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Rhodosporidium infirmo-miniatum Y1569, Rhodotorula aurantiaca Y985, Rhodotorula glutinis Y77, Rhodotorula lactosa Y2770, Rhodotorula minuta Y2777, Rhodotorula rubra Y1193, Saccharomyces cerevisiae Y1127, Saccharomyces kluyveri Y2559, Sporobolomyces holsaticus Y991, Sporobolomyces roseus Y987, Tilletiopsis washingtonensis Y1650, Torulopsis apicola Y566, Tremella foliacea Y1624, and Tremella mesenterica Y1625.
The results evidence that strains Bullera alba Y1581, Cryptococcus laurentii Y227, Cryptococcus macerans Y2763, Cystofilobasidium capitatum Y1852, Dioszegia hungarica Y3208, Dioszegia sp. Y3320, Geotrichum klebahnii Y3053, Phaffia rhodozyma Y1666, Rhodosporidium capitatum Y1567, Rhodotorula aurantiaca Y985, Rhodotorula minuta Y2777, Sporobolomyces holsaticus Y991, and Sporobolomyces roseus Y987 contain no pal gene and thus were excluded from further study.
Phylogenetic analysis of pal gene sequences in strains under study and reference strains from the GenBank database of genetic sequences (Table 1) was performed.
Upon multiple alignment of the sequences (CLUSTAL Omega) and their editing (intron excision), they were translated using the BioEdit 7.0.0 software. For further analysis, testing of various models of amino acid substitutions was performed with a ProtTest 3 service.
Structure of the PAL protein of Rhodosporidium toruloides was obtained from the PDB bank (1T6P, DOI:10.2210/pdb1t6p/pdb) in the PyMOL Molecular Graphics System, version 1.5.0.4 (Schrodinger, LLC). As follows from Fig. 2, the fragment lies in the internal part of the molecule and includes the region of a beta-sheet, two turns, and several alpha-helix regions.
Fig. 2. Structure of PAL protein from Rhodosporidium toruloides (DOI:10.2210/pdbH6p/pdbi. Green color indicates amino acids 398-472 (numbers are given according to the GI:3294 sequence) based on the results of alignment with the fragments under study in Clustal Omega software.
For a phylogenetic analysis in the PHYLIP 3.69 software package, 1000 bootstrap replicas of amino acid alignment were created, then the distances between the sequences (JTT substitution model, gamma = 1.78, based on a previously chosen optimal model) were calculated and the trees were built by a neighbor-joining method (NJ). The PAL sequence of Arabidopsis thaliana was used as an out-group. Figure 3 presents the phylogenetic consensus tree; joining was performed according to the 50% majority rule.
Also, MrBayes 3.2 software was used to built Bayes trees (based on a previously chosen model, Jones+G template of amino acid substitutions). The number of mcmc iterations was 600000. Figure 4 presents the phylogenetic tree with the possibility of node formation noted in the nodes. Again, the PAL sequence of Arabidopsis thaliana was used as an outgroup.
Based on the analysis of phylogenetic trees one may conclude that the studied sequences are divided into two clades. Subgroup Ia includes the following families: Aureobasidium, Candida, Cystofilobasidium, Debaryomyces, Phaffia, Puccinia, Rhodosporidium, Rhodotorula, Saccharomyces, Tilletiopsis, and Tremella. Clade II includes the following families: Aspergillus, Chaetomium, Letharia, Magnaporthe, Neosartorya, Neurospora, Penicillium, Phaeosphaeria, Talaromyces, and Uncinocarpus.
Upon the analysis of the tree obtained with Bayesian statistics one may note that the sequences also rather reliably divided into two clades, and the first one (lower clade) corresponds to subgroup Ia, while the second (upper) one corresponds to subgoup Ib and clade II (Figs. 3 and 4).
The template of identity of nucleotide and amino acid sequences (Tables 6 and 7) was created for some of the investigated strains under study (all sequences obtained in the current study, as well as Arabidopsis thaliana, Aspergillus niger, Neurospora crassa, Puccinia graminis, Rhodosporidium toruloides, Rhodotorula graminis, and Rhodotorula mucilaginosa sequences) using the CLUSTALW software.
Analysis of the data presented in Tables 5 and 6 evidences that the rate of identity of both nucleotide and amino acid sequences inside a clade is very high and makes up to 93-97% for some species.
A logo-diagram (Fig. 5) was designed for the Ia subgroup demonstrating the fragment is rather conserved.
Therefore, using the phylogenetic analysis the investigated gene has been shown to belong to the pal family, and the affinity of the sequences was evaluated.
The performed comparative analysis of sequences from genomes of pigmented yeasts with completely characterized genes encoding L-phenylalanine ammonia-lyase amplified and sequenced using the developed pair of primers demonstrated undoubtedly the sequences belong to the pal genes.
Therefore, we developed a universal primer system revealing genes encoding L-phenylalanine ammonia-lyase.
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Saccharomyces cerevisiae Candida apicola Tremella mesenterica Phaffia rhodozyma Candida glabrata Rhodosporidium infirmo-miniatum Debaryomyces robertsiae Cystofilobasidium capitatum Aureobasidium pullulans Candida maltosa Saccharomyces kluyveri Tilletiopsis washingtonensis Rhodotorula mucilaginosa Tremella foliacea Debaryomyces castellii Rhodosporidium diobovatum Rhodotorula rubra Rhodotorula glutinis Rhodosporidium toruloides Rhodotorula graminis Puccinia graminis f. sp. tritici Laccaria bicolor Coprinopsis cinerea okayama Tricholoma matsutake Pleurotus eryngii Amanita muscaria Ustilago maydis Agaricus bisporus Penicillium marneffei Talaromyces stipitatus Aspergillus oryzae Aspergillus flavus Neosartorya fischeri Aspergillus fumigatus Aspergillus clavatus Uncinocarpus reesii Letharia vulpina Magnaporthe oryzae Phaeosphaeria nodorum Aspergillus niger Aspergillus terreus Chaetomium globosum Neurospora crassa Arabidopsis thaliana
Fig. 3. Consensus tree designed with the PHYLIP 3.69 software using the data on PAL amino acid sequences obtained (trees were built using the neighbor-joining method, JTT substitution model, gamma = 1.78). In the nodes, the percent of bootstrap support is indicated for 1000 replicas. Bold font indicates the PAL sequences obtained in the work, and light font, sequences from NCBI databases.
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- Arabidopsis thaliana Aspergillus niger Aspergillus terreus Talaromyces stipitatus Penicillium marneffei • Uncinocarpus reesii Aspergillus clavatus Neosartorya fischeri Aspergillus fumigatus Aspergillus oryzae Aspergillus flavus - Letharia vulpina Phaeosphaeria nodorum Magnaporthe oryzae
□ Chaetomium globosum Neurospora crassa
---Ustilago maydis
---Amanita muscaria
---Coprinopsis cinerea okayama
---Laccaria bicolor
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Tricholoma matsutakee Pleurotus eryngii strain — Agaricus bisporus Phaffia rhodozyma Tremella mesenterica Candida glabrata Rhodosporidium infirmo-miniatum Cystofilobasidium capitatum Debaryomyces robertsiae ■ Saccharomyces cerevisiae
— Saccharomyces kluyveri
— Candida apicola
— Candida maltosa
—-Aureobasidium pullulans --Tilletiopsis washingtonensis
— Debaryomyces castellii
— Tremella foliacea
—-Rhodotorula mucilaginosa --Rhodotorula rubra
--Rhodotorula glutinis
— Rhodosporidium diobovatum
---Rhodosporidium toruloides
---Rhodotorula graminis
----Puccinia graminis f. sp. tritici
Fig. 4. Bayesian tree built using the MrBayes 3.2 software and the data on the obtained amino acid sequences of PAL (Jones+G substitution model). Node formation possibilities are indicated with the figures. Black letters indicate the sequences obtained in the work, and gray color, sequences from NCBI databases.
Fig. 5. Logo diagram of the PAL fragment, clade I.
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Table 5. Identity percent of the pal gene fragment nucleotide sequences in some species
Aspergillus niger 60
A ureobasidium pulhilans 23 24
Candida apicola 25 22 96 |
Candida glabrata 15 22 93 93 |
Candida maltosa 23 22 96 97 93 |
Cystofilobasidium capitatum 20 22 95 96 93 94 |
Debaryomyces castellii 26 25 90 86 92 93 JU
Debaryomyces robertsiae 18 22 95 96 92 94 96 95
Neurospora crassa 52 62 21 21 16 21 18 22 18
Phaffia rhodozvma 9 9 76 77 80 78 77 65 77 8
Puccinia graminis f. sp. tritici 52 59 20 19 19 23 22 30 19 59 16
Rhodosporidium diobovatum 37 40 87 84 91 92 95 97 94 33 59 43
Rhodosporidium infirmo-miniatum 18 22 95 96 94 95 96 96 96 19 78 18 94
Rhodosporidium toruloides 12 26 71 69 71 73 72 71 72 20 55 27 72 73
Rhodotorula glutinis 32 35 86 83 89 90 92 94 91 31 58 38 99 92 62
Rhodotorula graminis 43 47 35 34 32 34 34 41 34 49 21 57 58 33 43 50
Rhodotorula mucilaginosa 23 22 88 86 83 88 86 87 86 20 65 27 89 86 75 86 38
Rhodotorula rubra 29 34 80 77 82 84 85 88 85 27 51 35 89 85 72 87 48 97
Saccharomyces cerevisiae 16 23 91 90 94 91 95 95 96 21 74 27 95 95 70 91 39 85 86 |
Saccharomyces kluweri 20 22 90 90 87 91 89 92 88 18 73 22 93 89 72 91 33 83 84 88
Tilletiopsis washingtonensis 20 24 95 94 93 94 95 94 96 20 76 24 91 96 71 89 36 86 84 93 90
Tremella foliacea 23 23 95 94 92 94 95 97 95 18 76 24 97 95 73 93 38 87 87 95 90 96
Tremella mesenterica 17 22 84 82 82 83 84 78 82 16 67 19 79 84 62 77 32 76 71 82 80 84 83
Arabidopsis thaliana Aspergillus niger Aureobasidium pulhilans Candida apicola Candida glabrata Candida maltosa Cystofilobasidium capitatum Debaryomyces castellii Debaryomyces robertsiae Neurospora crassa Phaffia rhodozvma Puccinia graminis f. sp. tritici Rhodosporidium diobovatum Rhodosporidium infirmo-miniatum Rhodosporidium toruloides Rhodotorula glutinis Rhodotorula sraminis Rhodotorula mucilaginosa Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces kluweri Tilletiopsis washingtonensis Tremella foliacea
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Table 6. Identity percent of the pal gene fragment amino acid sequences in some species
Aspergillus niger 41
A ureobasidium pulhilans 40 41
Candida apicola 38 35 92
Candida glabrata 30 36 86 85
Candida maltosa 39 39 91 94 87
Cystofilobasidium capitatum 41 39 88 92 82 89
Debaryomyces castellii 43 43 92 84 84 90 87
Debaryomyces robertsiae 38 38 90 91 86 87 93 90
Neurospora crassa 43 53 41 40 35 41 43 44 41
Phaffia rhodozvma 26 26 63 63 63 60 65 60 65 31
Puccinia graminis f. sp. tritici 46 46 45 44 45 50 49 50 45 49 29
Rhodosporidium diobovatum 45 41 91 86 86 91 93 95 93 43 53 50
Rhodosporidium infirmo-miniatum 38 38 87 91 86 90 90 92 91 41 58 47 86
Rhodosporidium toruloides 47 47 73 71 67 73 72 80 69 50 51 57 84 71
Rhodotorula glutinis 47 39 91 87 85 91 93 95 91 43 53 52 100 87 85
Rhodotorula graminis 50 47 68 65 60 67 65 70 64 46 48 58 78 63 79 79
Rhodotorula mucilaginosa 42 42 94 92 84 94 90 96 89 43 63 52 100 89 79 100 72
Rhodotorula rubra 44 40 87 83 81 87 88 90 87 44 60 51 100 83 81 100 77 94
Saccharomyces cerevisiae 38 39 94 92 89 87 93 92 94 42 63 46 93 91 72 93 67 91 88
Saccharomyces kluweri 40 37 83 85 83 92 87 85 85 40 63 46 89 87 77 89 66 88 85 83
Tilletiopsis washingtonensis 41 42 93 88 86 90 90 95 91 43 63 49 93 91 75 93 68 94 88 94 88
Tremella foliacea 40 39 90 86 86 91 91 93 95 42 65 49 97 90 77 95 70 96 90 93 87 95
Tremella mesenterica 27 28 72 69 71 66 72 69 71 31 58 33 82 69 59 81 50 71 74 75 68 72 68
Arabidopsis thaliana Aspergillus niger Aureobasidium pulhilans Candida apicola v?vjqvj£ vpipuvj Candida maltosa Cystofilobasidiu m capitatum Debaryomyces castellii Debaryomyces robertsiae Neurospora crassa Phaffia rhodozvma Puccinia graminis f. sp. tritici Rhodosporidium diobovatum Rhodosporidium infirmo-miniatum Rhodosporidium toruloides Rhodotorula glutinis Rhodotorula sraminis Rhodotorula mucilaginosa Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces kluweri Tilletiopsis washingtonensis Tremella foliacea
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Study of the Specific Features of Phenotype and Biochemical Properties of the Selected Cultures
Phenotypic characteristics of the selected pigmented yeasts were judged by the combination of micro- and macromorphological traits (the former are studied with a microscope and the latter, visually). Micromorphology includes the features characterizing individual vegetative cells (shape, size), as well as the types of vegetative or asexual reproduction and the structures formed in the process. Macromorphology joins the culture features characterizing culture growth on dense (following the streak or in the form of a giant colony) or in liquid medium.
On a complete yeast medium (g/cm3 distilled water: peptone, 10; yeast extract, 5; glucose, 20; agar, 20), yeasts form colonies of intermediate size and white or creamy color, colony surface is smooth, dim, elevated by a cone, colony edges are even or slightly wavy.
Description of the isolated yeast strains was per-Table 7. Phenotypic traits of pigmented yeasts
formed according to a standard scheme. Shape and size of cells were described and determined in cultures of different ages on dense and liquid media. The first examination fnd measurement of cell size was performed in 2-3-day cultures grown at 25-28°С. Then, the cultures were left at room temperature (17-18°С) and described again after 4 weeks.
To determine cell size, length and width were measured with a micrometer in at least 20 cells, and the extreme values were indicated. Cells of isolated yeasts were found to be of round, round-like, oval, and cylindrical shape.
Size of mature yeast cells varied in different species from 1.0 to 8.0 pm in width and reached 17 pm and more in length in case of elongated cells. The results are presented in Table 7.
Further studies were aimed at determination of L-phenylalanine ammonia-lyase activity in the selected pigmented yeasts. The results of the investigation are presented in Table 8.
Strain Size, pm Shape Edge outline Relief Surface Color
Candida glabrata Y2813 2.5 x 6.0 oval uneven edges smooth dim creamy
Aureobasidium pullulans Y863 8.0 x 6.0 ellipse-like uneven edges wrinkled dim creamy
Tremella foliacea Y1624 2.0 x 6.5 round uneven edges smooth shiny creamy
Phaffia rhodozyma Y1668 3.8 x 10.0 round-to- oval uneven edges smooth dim red orange
Rhodotorula lactose Y2770 2.5 x 8.0 oval-to- elongated uneven edges smooth shiny red pink
Rhodosporidium diobovatum Y1565 1.0 x 9.0 round-to- oval uneven edges wrinkled dim red pink
Rhodotorula rubra Y1193 2.3 x 6.5 oval-to- elongated even edges smooth shiny red pink
Tremella mesenterica Y1625 2.0 x 8.5 round-to- oval uneven edges smooth shiny creamy
Debaromyces castellii Y 968 3.8 x 8.5 round-to- oval uneven edges wrinkled dim white
Candida maltose Y242 2.0 x 7.0 round-to- cylindrical even edges smooth shiny creamy
Rhodosporidium capitatum Y1567 1.0 x 14.0 round-to- cylindrical even edges smooth dim pink orange
Saccharomyces kluyveri Y2559 5.0 x 12.0 round-to- cylindrical even edges smooth shiny light creamy
Bullera alba Y 1581 2.0 x 6.5 oval uneven edges smooth dim creamy
Rhodotorula glutinis Y77 2.3 x 10.0 round-to- oval even edges smooth dim red pink
Rhodosporidium infirmominia-tum Y1569 1.0 x 10.0 round-to- oval even edges smooth dim red pink
Debaryomyces robertsiae Y3392 2.8 x 8.0 round-to- oval uneven edges wrinkled dim white
Saccharomyces cerevisiae Y1127 5.0 x 12 round-to- cylindrical even edges smooth shiny light creamy
Tilletiopsis washingtonensis Y1650 1.0 x 17.0 round-to- cylindrical uneven edges wrinkled dim creamy
Torulopsis apicola Y566 2.0 x 6.0 round-to- oval even edges smooth shiny yellowish- creamy
Note:. Width x length.
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Table 8. L-phenylalanine ammonia-lyase activity in selected pigmented yeasts
No. Yeast L-phenylalanine ammonia-lyase activity, U/mg protein
1 Aureobasidium pullulans Y863 0.038
2 Bullera alba Y1581 0.010
3 Bullera piricola Y1577 0.008
4 Candida glabrata Y2813 0.007
5 Candida maltosa Y242 0.012
6 Cryptococcus laurentii Y227 0.010
7 Cryptococcus macerans Y2763 0.008
8 Cystofilobasidium capitatum Y1852 0.010
9 Debaryomyces castellii Y968 0.007
10 Debaryomyces robertsiae Y3392 0.012
11 Dioszegia hungarica Y3208 0.010
12 Dioszegia sp. Y3320 0.008
13 Geotrichum klebahnii Y3053 0.009
14 Phaffia rhodozyma Y1668 0.009
15 Rhodosporidium diobovatum Y1565 0.022
16 Rhodosporidium infirmominiatum Y1569 0.019
17 Rhodotorula glutinis Y77 0.008
18 Rhodotorula lactosa Y2770 0.049
19 Rhodotorula rubra Y1193 0.015
20 Saccharomyces cerevisiae Y1127 0.016
21 Saccharomyces kluyveri Y2559 0.010
22 Sporobolomyces holsaticus Y991 0.009
23 Tilletiopsis washingtonensis Y1650 0.017
24 Torulopsis apicola Y566 0.011
25 Tremella foliacea Y1624 0.019
26 Tremella mesenterica Y1625 0.010
The data presented in Table 8 evidence that the highest activity was exhibited by the strains
Aureobasidium pullulans Y863, Rhodosporidium infirmominiatum Y1569, Candida glabrata Y2813, Candida maltose Y242, Debaryomyces robertsiae Y3392, Rhodosporidium diobovatum Y1565, Rhodotorula lactose Y2770, Saccharomyces cerevisiae Y1127, Tilletiopsis washingtonensis Y1650, Torulopsis apicola Y566, Tremella foliacea Y1624, Rhodotorula rubra Y1193, and Debaryomyces castellii Y968, which allows to recommend them for further studies aimed at the development of an enzyme preparation of L-phenylalanine ammonia-lyase.
Therefore, we analyzed the DNA sequence encoding the synthesis of L-phenylalanine ammonia-lyase (PAL) to create universal primers complementary
to conserved regions of the pal gene. The pal gene fragment was amplified in the organisms under study. Nucleotide sequence of the pal gene in microorganisms possessing L-phenylalanine ammonia-lyase activity was determined by DNA sequencing. The sequence was compared with the relevant sequences of known species. Phenotypic features and biochemical properties of the selected cultures were investigated.
Acknowledgments
Authors are grateful to the Sintol company (Moscow, Russia) for sequencing of the indicated yeast strains.
The paper was prepared in frames of the target federal program “Scientific and Academic Stuff for Innovative Russia” in the years 2009-2013 (grant agreement no 14.V37.21.0565, 10.08.2012).
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