The analysis of the polyamine oxidase genes in the methylotrophic yeast Komagataella phaffii

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INTRODUCTION

Polyamines are a group of aliphatic polycations which are present in prokaryotes and eukaryotes. Putrescine, spermidine and spermine are the most commonly found polyamines in higher eukaryotes, including fungi. The functions of polyamines are determined by their chemical structure. Polyamines are positively charged at physiological pH, and due to electrostatic and hydrophobic interactions they can act as ligands, binding to DNA, RNA, proteins, phospholipids and nucleoside triphosphates [1, 2]. Polyamines are considered to be involved in the regulation of gene expression by altering the DNA structure, modulating the signal pathways or binding to the transcription factors. Therefore, polyamines are redundant in all living organisms. Polyamine deficiency leads to the cessation of cell growth, however excessive accumulation of these compounds can be cytotoxic, and therefore strict regulation of polyamine intracellular levels is needed [2, 3]. The present interest in polyamines is connected with their possible use as antiproliferative compounds.

The intracellular level of polyamines is controlled by a complex of biosynthetic (ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine and spermine synthases) and catabolic (spermidine/spermine acetyltransferase, FAD-containing polyamine oxidase and copper-containing diamine oxidase) enzymes [2]. Polyamine oxidase is likely to be of particular interest, since it catalyzes the convertion of spermine to spermidine and 3-aminopropanal. The oxidation of 3-aminopropanal is required for S. cerevisiae for the biosynthesis of pantothenic acid [4], while spermidine is involved in the reaction of hipusination (the essential modification of the translation factor eIF-5A) [5].

The phylogenetic analysis of polyamine oxidases was performed for animals and plants. It has been discovered that during the evolution of animals, the ancestral gene of polyamine oxidase (PAO) was duplicated, and as a result, two protein paralogs of vertebrates emerged, encoded by SMO and APAO genes [6]. Additionally, the genome of Arabidopsis thaliana encodes for at least five polyamine oxidases. The analysis of the substrate specificity revealed that polyamine oxidase AtPAO13 oxidizes spermidine and spermine producing H₂O₂. Equally, it has been shown that polyamine oxidases of A. thaliana AtPAO2, AtPAO3 and AtPAO4 are localized in peroxisomes. One of the animal PAOs carries a peroxisomal targeting signal and is likely to be localized in these organelles.

In fungi polyamines regulate a wide range of biological phenomena: dimorphism, spore germination and the formation of appresorium. In several cases they control the virulence of fungal pathogens of animals and plants [8]. FAD – dependent (flavin adenine dinucleotide) polyamine oxidase Fms1p of Saccharomyces cerevisiae is studied in detail [9], and shown to be involved in β-alanine and pantothenate biosynthesis. Yeast mutants with FMS1 deletion are unable to grow on media without pantothenic acid or β-alanine, while the overexpression of FMS1 gene under the control of ADH1 promoter leads to the secretion of pantothenic acid into the growth media [4]. At the same time, the number of polyamine oxidase genes varies among other members of the Saccharomycotina subdivision, and the correlation between the gene copy number of polyamine oxidase and the metabolism rate is not clear.

This work analyzes the orthologs of polyamine oxidases among the spp. of the Saccharomycotina subdivision. In this report we show that the methylotrophic yeasts Komagataella phaffii (Pichia pastoris) has two polyamine oxidase genes – KpFMS1 and KpFMS2. The regulation of these genes have been studied.

MATERIALS AND METHODS

Comparison of the polyamine oxidase amino acid sequences

The orthologs of the S. cerevisiae Fms1 protein were found among the spp. of the Saccharomycotina group, using BLASTp algorithm with standard parameters [10]. The multiple alignment of the polyamine oxidase sequences was performed in MEGA X computer software [11]. The found sequences were aligned in CLUSTAL W [12]. The Maximum Likelihood (ML) method was used for the phylogenetic tree construction. The most appropriate model for this method was LG+G [13]. The construction of the ML tree was carried out using LG+G model with 500 bootstrap iterations. The final tree was rooted with the Neurospora crassa outgroup, and the tree was visualised in FigTree v1.4.3(http://influenza.bio.ed.ac.uk/software/Figtree/).

Primers

Primers used are listed in Table 1.

 

Plasmids

pAL2-T (Evrogen, Russia) and pPIC9-AOX1-PHO5 [14] vectors were used in this work.

Strains

Yeast strains K. phaffii 4-GS115 (his4 phox) and tr2-4-GS115 (phox PAOX1-PHO5) [14], and the bacterial strain E. coli DH5α, (fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17) were used in this work.

Media and culture conditions

For the cultivation of the yeast strains the following media were used. YEPD: 2% glucose, 2% peptone, 1% yeast extract, 2.4% agar; MPO (MPhO): 0.73 M MgSO₄ ∙ 7H₂O, 0.18 mM CaCl₂, 20 mM Sodium Citrate Buffer (pH 4.6), vitamins, trace elements. Сarbon source (glycerol, methanol) – 1%, nitrogen source (ammonium sulfate) – 0.2%. For the cultivation of bacteria LB medium was used: 1% tryptone, 0.5% yeast extract, 170 mM NaCl, benzylpenicillin – 5 ∙ 10⁵ units of activity/l. The K. phaffii strains were cultivated at 30 °C, the E.coli – at 37 °C.

Construction of pPIC9-PKpFMS1-PHO5 and pPIC9- PKpFMS2-PHO5 plasmids

To construct pPIC9-PKpFMS1-PHO5 and pPIC9-PKpFMS2-PHO5 plasmids, polymerase chain reaction (PCR) was carried out with primers designed to specifically anneal to the promoter regions of KpFMS1 (KpFMS1F and KpFMS1R) and KpFMS2 (KpFMS2F and KpFMS2R) genes, with the chromosomal DNA of the K. phaffii 4-GS115 strain used as a template. The extraction of the chromosomal DNA from the yeast cells was carried out as described in [15]. The promoter sequences were cloned into pPIC9-PHO5 vector [14] using AatII and BamHI restriction sites, thus the promoter of alcohol oxidase gene (AOX1) was replaced by the promoters of the polyamine oxidase genes KpFMS1 and KpFMS2. The transformation of bacteria was carried out according to [16]. As a result, the constructed plasmids contained the sequence of acid phosphatase (AP) PHO5 reporter gene from S. cerevisiae under the control of KpFMS1 and KpFMS2 gene promoters. Moreover, these plasmids hold HIS4 gene as a selective marker.

The structure of the final plasmids pPIC9-PKpFMS1-PHO5 and pPIC9-PKpFMS2-PHO5 was checked using PCR and restriction analysis.

The engineering of the PFMS1-4-GS115 and PFMS2-4-GS115 strains

The 4-GS115 strain was transformed with pPIC9-PKpFMS1-PHO5 and pPIC9-PKpFMS2-PHO5 plasmids. To that end, the plasmids underwent linearization by StuI restriction endonuclease, and the yeast cells were transformed via electroporation [17]. During electroporation, the genetic construction was incorporated into K. phaffii genome. The selection of transformants was performed by the restoration of histidine prototrophy. To analyze the integration of the plasmid into the genome, chromosomal DNA was extracted from the transformants, and PCR was carried out using PAO and PHO5 specific primers (KpFMS1F and PHO5R for PFMS1-4-GS115; KpFMS2F and PHO5R for PFMS2-4-GS115).

Molecular methods

The hydrolysis of DNA was performed by BamHI, AatII and StuI restriction endonucleases following the manufacturer’s recommendations (Thermo Fisher Scientific Inc., USA). The vectors were dephosphorylated using FastAP phosphatase (Thermo Fisher Scientific Inc., USA). The purification of DNA from agarose gels and reaction mixtures was accomplished with Cleanup Standard kits (Evrogen, Russia). The ligation of DNA fragments was performed by T4 DNA ligase (Evrogen, Russia). The plasmids were extracted using Plasmid Miniprep kits (Evrogen, Russia). To carry out PCR, Encyclo Plus PCR kits were used (Evrogen, Russia). The electrophoresis of DNA fragments was performed in the 0.7% agarose gel in TAE buffer [18].

Qualitative evaluation of the acid phosphatase activity

Paper filters were soaked in a solution containing α- naphthyl phosphate as the substrate and Fast Blue B Salt as a dye, diluted in 0.1 M citrate buffer pH 4.6 to 2 mg/ml. The wet filters were then placed on the surface of the medium containing grown yeast colonies. After 10 minutes the colony staining intensity was measured [19].

RESULTS

1. Bioinformatic analysis of Saccharomycotina yeast proteomic data

At the first stage of our work we studied the prevalence of polyamine oxidases among major representatives of Saccharomycotina subdivision (Ascomycota division). We searched for homologous proteins in the proteomes of 22 yeast species belonging to different families of this division and in one proteome of Neurospora crassa fungus (the representative of the Pezizomycotina subdivision, Ascomycota division). These data were obtained by means of BLAST analysis using the amino acid sequence of S. cerevisiae polyamine oxidase Fms1p as a template [9]. A similar approach was applied earlier in the search of polyamine oxidases in phytopathogenic fungi [8]. The results are shown in Fig. 1.

 

Fig. 1. The prevalence of polyamine oxidases among the main representatives of the Saccharomycotina subdivision. Phylogenetic relationships are shown based on the results obtained in [21]. WGD refers to the whole-genomic duplication that the ancestors of S. cerevisiae and related species underwent [28]

 

We identified that polyamine oxidases and corresponding genes are present in all investigated species from different families of the Saccharomycotina subdivision. Furthermore, the number of polyamine oxidase proteins and corresponding genes varies according to the systematic position of the yeast species: 1) the major representatives of Ascoideaceae, Phaffomycetaceae, Saccharomycetaceae families possess only one gene, 2) the representatives of Debaryomycetaceae, Metschnikowiaceae families and Yarrowia clade have two genes, 3) the representatives of Pichiaceae family possess three genes. An exception to this family is the yeast species Komagataella phaffii (better known as Pichia pastoris), who contains two genes. This yeast species is a representative of Komagataella clade regarded as the paraphyletic family Pichiaceae in the report [20].

Carrying out phylogenetic analysis with the use of the polyamine oxidase gene sequences was complicated due to the large differences among nucleotide sequences of the found genes. Hence, multiple comparisons of polyamine oxidase amino acid sequences were performed for all the investigated yeast species (Fig. 2).

 

Fig. 2. The results of multiple comparisons of polyamine oxidases amino acid sequences from the studied yeast species. The table shows the PTS1 sequences at the C-terminus of polyamine oxidases, which provide the peroxisomal localization of proteins. The underlined amino acids differ from the consensus sequence known for S. cerevisiae [22]

 

The results enabled us to divide the amino acid sequences of polyamine oxidases into groups (PAO) based on their similarity. PAO1 group contains proteins found in representatives of the Ascoideaceae, Phaffomycetaceae, Saccharomycetaceae families, with only one polyamine oxidase gene identified in their proteomes. Whereas in PAO2.1 and PAO2.2 groups comprising proteins discovered in the representatives of the Metschnikowiaceae family, two polyamine oxidase genes were recognized. Finally, in PAO3.1, PAO3.2 and PAO3.3 groups comprising proteins found in the representatives of the Pichiaceae family, three genes were identified. The Babjeviella inositovora proteins (Debaryomycetaceae, two genes) also belong to these groups (PAO3.1 and PAO3.2). The results of the sequence multiple alignments within PAO groups generally reflect the phylogenetic relationships in corresponding families of the Saccharomycotina subdivision.

2. Bioinformatic analysis of the polyamine oxidase subcellular localization

A highly toxic by-product hydrogen peroxide is generated during reactions mediated by polyamine oxidases. Hence, several polyamine oxidases in plants and animals are directed to peroxisomes [7]. For all the investigated polyamine oxidases, we searched for the signal sequences that provide their transport to various cell organelles, primarily to peroxisomes. Two main types of signals are known to implement protein targeting to peroxisomes – PTS1 and PTS2 (Peroxisomal Targeting Signals). PTS1 is prevalent, it is found in 95% of the peroxisomal matrix proteins. Initially, the PTS1 signal was defined as a sequence comprising three amino acids at the C-terminus of a protein – SKL. However, it was subsequently shown that this signal can vary and thus it is a consensus sequence [21]. The polyamine oxidases were analyzed for having peroxisomal targeting signals, and the results are shown in Fig.2.

We report that two groups PAO2.2 and PAO3.2 can be distinguished among the studied polyamine oxidases, and their representatives often (in 6 out of 9 cases) possess a consensus C-terminal PTS1 signal. Meanwhile, three proteins have a sequence at the C-terminal end, not corresponding to the consensus signal but highly comparable. The polyamine oxidase sequences were analyzed in subcellular localization predictor server DeeLoc-1.0 (http://www.cbs.dtu.dk/services/DeepLoc/). In order to predict subcellular protein localization, this program uses machine learning methods instead of searching for known signal sequences in databases [22]. Aided by this algorithm we identified that the most probable subcellular localization outcome of K. phaffii polyamine oxidase XP_002494272.1 (KpFMS2 gene) is the peroxisomes.

K. phaffii yeasts studied in the current work are methylotrophic microorganisms, i. e. they are capable of utilizing methanol as the only carbon and energy source. The first steps in methanol metabolic pathway occur in peroxisomes, which is also connected with the generation of hydrogen peroxide by alcohol oxidases during methanol oxidation to formaldehyde [23]. The analysis of K. phaffii genome has shown two unlinked polyamine oxidase genes KpFMS1 and KpFMS2, with KpFMS2 located close to alcohol oxidase 1 gene (AOX1). Besides, KpFMS2 and AOX1 genes are arranged head to head, and their promoter regions may partially share a sequence (Fig. 3), feasibly affecting the regulation of KpFMS2 transcription.

 

Fig. 3. The arrangement of KpFMS2 and AOX1 K. phaffii genes. The binding sites of the transcription factors Mxr1p and Nrg1p (the main regulators of AOX1 gene) are shown

 

3. Comparative study of the KpFMS1 and KpFMS2 gene expression in K. phaffii yeast

In order to study the regulation of the activity of polyamine oxidase gene promoter, we developed PFMS1-4-GS115 and PFMS2-4-GS115 strains, each containing the reporter gene sequence of acid phosphatase PHO5 under the control of polyamine oxidase gene promoters. To compare KpFMS1 and AOX1 gene promoter activities, we used tr2-4-GS115 strain [14], which contains PHO5 gene under the control of AOX1 gene promoter. Serial dilutions of the strains were prepared and plated on agar media with different carbon and nitrogen sources. Glycerol and methanol were used as the sole carbon source. Ammonium sulfate was used as a nitrogen source. Besides, YEPD medium was used, containing glucose, yeast extract and peptone. The results of the qualitative analysis of the reporter gene PHO5 activity in PFMS1-4-GS115, PFMS2-4-GS115 and tr2-4-GS115 strains are shown in Fig. 4.

 

Fig. 4. The activity of reporter acid phosphatase synthesized by K. phaffii PFMS1-4-GS115, PFMS2-4-GS115 and tr2-4-GS115 strains during their growth on the media with different carbon and nitrogen sources

 

The activity of KpFMS2 gene promoter was demonstrated on YEPD medium, where glucose was used as a carbon source, and the nitrogen source was a mixture of amino acids and oligopeptides. We did not observe the activity of KpFMS1 gene promoter in these conditions, and as it was expected [24], AOX1 gene was not expressed.

Polyamine oxidase genes promoters are not active in media with glycerol as a carbon source. The expression of AOX1 gene was repressed by glycerol in the studied conditions.

KpFMS1 and AOX1 gene promoters are active in media with methanol as a carbon source. The activity of KpFMS1 gene promoter was not observed.

DISCUSSION

It is established that duplications play a significant role in the evolution of living organisms. Duplications create additional copies of the genetic material and contribute to genome diversity, and the fate of duplicated genes can vary. Mutations can accumulate whether in a coding or a regulatory region. As a result, duplicated genes can undergo the following processes throughout evolution: 1) conservation, during which both copies maintain functions of an ancestral gene, 2) neofunctionalization, during which one gene develops a new function, while another preserves ancestral functions, 3) subfunctionalization, during which both copies acquire different functions and work together in order to compensate functions of an ancestral gene, and 4) specialization, when two copies gain different functions, and their common function is not similar to the ancestral one (in other words, it is a combination of neofunctionalization and subfunctionalization processes) [25, 26]. Moreover, structural genes can become a part of other regulons due to the changes in regulatory regions, developing the capability of responding to new signals via interactions with new regulatory factors.

In the current work, for the first time, we performed a bioinformatic analysis of the polyamine oxidase distribution among major representatives of the Saccharomycotina subdivision (Ascomycota division). We showed that all the studied species have polyamine oxidases and corresponding genes; however, their copy number varies. Besides, we demonstrated that the copy number correlates with the systematic position of the investigated yeast species. It is interesting that representatives of the Saccharomycetaceae family (S. cerevisiae yeast species) and the related Ascoideaceae, Phaffomycetaceae families contain a single polyamine oxidase gene. It should be noted that the ancestors of S. cerevisiae underwent the whole-genome duplication [27]. Accordingly, one of the two polyamine oxidase genes evolved after duplication in S. cerevisiae, and was subsequently lost, nevertheless, a single copy is sufficient to maintain the function.

Meanwhile, in other families of the Saccharomycotina subdivision representatives retained two or even three polyamine oxidase genes. The sequences of these genes diverged significantly, while the expressed proteins are supposed to have different subcellular localization. One polyamine oxidase (PAO2.2 and PAO3.2 groups) was found in most of the studied species (representatives of the Debaryomycetaceae, Metschnikowiaceae, Pichiaceae families and Yarrowia clade) containing a peroxisome targeting sequence. Polyamine oxidases from B. inositovora, Metschnikowia spp. and Ogataea parapolymorpha, (PAO2.2 and PAO3.2 groups), bear at their C-terminal end similar sequences to PTS1 of S. cerevisiae but not corresponding with consensus. Further analysis of polyamine oxidase subcellular localization will expand the set of peroxisomal targeting signals known for yeasts.

The differences discovered in the frequency of polyamine oxidase genes may be associated with peculiarities of metabolism in different yeast species. The study of the polyamine oxidase gene regulation in K. phaffii methylotrophic yeasts is of great interest in this regard. This yeast species is capable of using methanol as the only source of carbon and energy, and alcohol oxidase catalyzes the first chemical reaction of methanol metabolism. This enzyme functions within peroxisomes, since its activity is associated with the generation of the cytotoxic by-product hydrogen peroxide. Alcohol oxidase 1 gene is strictly regulated by the carbon source in the medium. AOX1 gene is repressed in media containing glucose and glycerol, while AOX1 promoter is induced in media with methanol [24]. The Mxr1p transcription factor is the main regulator of the AOX1 gene promoter activity [28].

In this work we showed that polyamine oxidase KpFMS2 gene of K. phaffii is located close to AOX1 gene, and they are arranged head to head. The binding sites of Mxr15’-CYCC-3’ protein are distributed throughout the region between the coding sequences of KpFMS2 and AOX1 genes on both strands (Fig. 3). Nrg1p protein acts as a repressor of AOX1 gene in K. phaffii in the presence of either glycerol or glucose in the media. The first region with which Nrg1p interacts is situated close to KpFMS2 coding sequence, while the second one is located close to AOX1 gene (Fig. 3). Therefore, the promoter regions of these genes may partially share a sequence, i. e. they may share similar elements of regulation.

In this work it was shown for the first time that KpFMS2 gene promoter (sharing a sequence with AOX1 gene promoter and providing transcription in the opposite direction) functions in media with methanol. The level of its activity was noticeably lower than of AOX1 gene promoter. KpFMS2 gene promoter, as well as AOX1 gene promoter, was inactive in media with glycerol as a source of carbon. However, at the same time KpFMS2 gene promoter was active in YEPD medium. In other words, unlike AOX1 gene promoter its activity was not suppressed by glucose in case of K. phaffii growth on this medium.

Thus, KpFMS2 gene appeared to be linked with AOX1 gene as a result of chromosomal rearrangements. While this gene preserves its own regulation features, its promoter is regulated similarily as AOX1 gene promoter and the other genes of the methanol metabolic pathway that form MUT-regulon. On the one hand, it can be a side effect of the remarkably high activity of AOX1 gene promoter. In fact, eukaryotic promoters can provide transcription of long noncoding RNAs [31]. On the other hand, the involvement of KpFMS2 gene into the MUT-regulon may be established during the course of evolution. The fact supporting this idea is that PAO3.3 group of polyamine oxidases including KpFMS2 protein is close to PAO3.2 group containing proteins with PTS1 signals. In spite of the fact that KpFMS2 protein does not possess such a sequence, its targeting to peroxisomes is predicted with means of DeepLoc-1.0 server. The MUT-regulon genes are expressed when K. phaffii is growing in methanol containing media during which, peroxisomes are actively formed and functional. The involvement of KpFMS2 gene into the MUT-regulon probably emerge to be beneficial and was selected throughout evolution.

The activity of KpFMS1 gene promoter was not observed in the studied conditions. It may be the consequence of the promoter complete inactivity or its putative activity under specific conditions. Further investigation of K.phaffii polyamine oxidase gene regulation and function is important both from a theoretical point of view in order to understand the evolution of duplicated genes, and practically, according to the immense significance of these genes and the MUT-regulon promoters for biotechnology.

Acknowledgements

This work was supported by RFBR № 18-34-00750 mol_a grant.

About the authors

Alina V. Ivanova

St. Petersburg State University

Email: alinalans@gmail.com

4^th year Student of the Department of Genetics and Biotechnology

Russian Federation, 7/9, Universitetskaya embankment, Saint-Petersburg, 199034

Anton V. Sidorin

St. Petersburg State University

Email: spacerocketpilot@gmail.com

Bachelor of Science (BSc) in the Genetics and Biotechnology Department

Russian Federation, 7/9, Universitetskaya embankment, Saint-Petersburg, 199034

Elena V. Sambuk

St. Petersburg State University

Email: esambuk@mail.ru
ORCID iD: 0000-0003-0837-0498
SPIN-code: 8281-8020
Scopus Author ID: 6603061322
ResearcherId: H-6895-2013

Doctor of Science, Professor of the Department of Genetics and Biotechnology

Russian Federation, 7/9, Universitetskaya embankment, Saint-Petersburg, 199034

Andrei M. Rumyantsev

St. Petersburg State University

Author for correspondence.
Email: rumyantsev-am@mail.ru
ORCID iD: 0000-0002-1744-3890
SPIN-code: 9335-1184
Scopus Author ID: 55370658800

PhD, Senior Researcher of the Department of Genetics and Biotechnology

Russian Federation, 7/9, Universitetskaya embankment, Saint-Petersburg, 199034

References

Supplementary files