Biotransformation Mechanism of Inorganic Selenium into Selenomethionine and Selenocysteine by Saccharomyces Boulardii: In Silico Study

The biosynthesis of inorganic selenium into seleno amino acids has been studied in recent years. Thus, it has been reported that Saccharomyces cerevisiae bioaccumulates selenium from the metabolism of inorganic selenium. Based on the studies conducted, several authors have proposed a biotransformation metabolism of selenate into selenomethionine or selenocysteine. However, the pathway in different yeast is unknown. Therefore, and given the relevance of Saccharomyces boulardii as probiotic yeast, this study aims to propose the pathway used by S. boulardii to biosynthesize inorganic selenium into organic species. A comparative in silico study was performed for Saccharomyces boulardii ASM141397V1 with the genome-scale metabolic model of Saccharomyces cerevisiae S288C. Orthologous genes were identied using BLASTp of NCBI. In addition, a circular representation was done using CIRCOS software. The metabolic pathway for the assimilation of selenium was proposed based on the results obtained


Introduction
Yeasts can permanently incorporate ions from the environment into their cell structure (Kieliszek and  On the other hand, Saccharomyces boulardii is a yeast originally isolated from lychee and is the only probiotic one approved for human consumption by the Food and Drug Administration (FDA) (McCullough et al. 1998; Moon et al. 2020). The regular use of Saccharomyces boulardii prevents and treats acute diarrhea associated to bacterial infections and other gastrointestinal disorders (Moon et al. 2020). In addition to studies on its probiotic properties (Czerucka et al. 2007), Saccharomyces boulardii has been reported to produce selenium nanoparticles (Bartosiak et al. 2019). Therefore, the production process of organic selenium by yeasts is considered a green technology (Bartosiak et al. 2019;Patel et al. 2013).
Despite the number of studies available, only a few explain the use of Saccharomyces boulardii to obtain selenomethionine (SeMet) and/or selenocysteine (SeCys). The capacity of this yeast to produce seleno amino acids represents an opportunity in biotechnology to obtain more bioavailable and less toxic selenium (Schrauzer 2000;Kitajima and Chiba 2013).
Studies have proven that the biosynthesis of selenium takes place through a pathway similar to that of sulfur. Selenium replaces sulfur and is incorporated into the cell as SeMet and/or SeCys (Kieliszek and Błażejak 2013; Bierla et al. 2013). Another mechanism proposed is transsulfuration through a non-speci c enzyme pathway yet to be documented (Ouerdane and Mester 2008). Even though these mechanisms exist, not all yeasts synthesize both seleno amino acids, such is the case of Saccharomyces cerevisiae BY4741 which does not synthesize methionine using inorganic sulfur (Ouerdane and Mester 2008). Therefore, some genes involved in enzyme coding for selenium synthesis in Saccharomyces cerevisiae are likely orthologous to those found in Saccharomyces boulardii. So, given that the genome of Saccharomyces cerevisiae is the most studied and best characterized among eukaryotes (Fisk et al. 2006), in this work we performed an in silico study to identify the probable orthologous genes involved in selenium biosynthesis by Saccharomyces boulardii (nom. inval.) ASM141397v1 to propose a pathway for sodium selenate biotransformation into SeMet and SeCys.

Materials And Methods
Identi cation of the aim The genetic comparison was carried out through an in silico study. Saccharomyces boulardii (nom. inval.) ASM141397v1 was used as study object. It was compared against the sequence of Saccharomyces cerevisiae s288c due to the genetic similarity between both yeasts. The process was carried out according to the models iMM904 (King et al. 2016) and Kegg (Ogata et al. 1999) for S. cerevisiae. The information on the genomes of the yeasts was obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/genome).

Bioinformatic search
To perform the search of the genes involved in the production of SeMet and SeCys, we used the Entrez in the NCBI database (https://www.ncbi.nlm.nih.gov/genome). We searched sequences of the genes reported in selenium metabolism for Saccharomyces cerevisiae s288c. The same procedure was carried out for Saccharomyces boulardii (nom. inval.) ASM141397v1. Sequences for the identi cation of orthologous genes were prepared.

Orthologous genes
With the information gathered, we searched the orthologous genes using the BLAST server at NCBI (Stephen et al. 1997). BLASTp program (protein-protein BLAST) was used to blast a protein sequence which became the input in FASTA format. In addition, we chose to collate the data exclusively with S. cerevisiae s288c to compare sequences and nd regions of local similarity between them (Bhagwat and Aravind 2007). A total number of 9361 amino acids were analyzed, and a circular scheme was created with Circos software (http://circos.ca/).

Metabolic pathway construction
According to the analysis of homologous genes found, a metabolic pathway was proposed for selenium assimilation and the subsequent production of SeMet and SeCys by Saccharomyces boulardii. The construction considered the proposals by Kieliszek et al. (2015) and Lazard et al. (2018) for S. cerevisiae.

Results And Discussion
Bioinformatic search According to Lazard et al. (2018), the genes involved in the biotransformation of selenate into SeMet and SeCys for Saccharomyces cerevisiae are those shown in Table 1. These genes reported were searched in the gene sequence for S. boulardii. Although the genes identi ed for S. boulardii are putatively named, the enzyme they encode for is the same in both yeasts.

Orthologous genes
Once the genes in S. boulardii were identi ed, a homology analysis was conducted to verify whether the genes between strains were orthologous (Stephen et al. 1997). Figure 1 shows

Discussion
The biotransformation of selenate into organic selenium starts with yeast detoxi cation by an excess of sodium selenate. The pathway through which this takes place is similar to that of sulfur metabolism for the production of sulfur-containing amino acids. In this pathway, selenium substitutes sulfur and is incorporated into the chemical structure of methionine and cysteine (Bierla et al. 2013).
Noting that biosynthesis starts with selenium detoxi cation and is carried out through a pathway similar to that of sulfur, we propose the beginning of absorption. Selenium could be absorbed in two different ways. The rst one is through sulfur ABC membrane transporters, which are encoded by operon cysAWTP and where transport for selenium ions uses energy from hydrolysis of bound ATP. The second system is through the transport of selenium using sulfate permeases (Kieliszek et al. 2015) encoded by AB282_00450 and AB282_03394. These enzymes transfer selenate through the plasma membrane from the exterior.
Once the selenate is in the interior, the biotransformation process starts with the activation of selenate. This process is carried out through a sequence of two reactions. In the rst one, the rest of the adenosylphosphoryl is transferred from ATP to selenate by the action of enzyme ATP sulfurylase encoded by AB282_02749. This produces adenylyl selenate, which is in turn phosphorylated to produce 3'phosphoadenylyl selenate through enzyme adenylyl-sulfate kinase (AB282_03058). Activated selenate is reduced to sul te to carry out SeMet and SeCys biosynthesis. First, enzyme 3'-phosphoadenylsulfate reductase (AB282_05395) reduces it to adenosine 3',5'-bisphosphate and free selenite, using reduced thioredoxin as substrate. Consecutively, the subunit alpha of assimilatory sul te reductase (AB282_01793), turns selenite into hydrogen selenide. Selenide is transformed into selenohomocysteine by the action of O-acetylserine-O-acetylhomoserine sulfhydrylase (AB282_03569).
The biosynthesis reaction of SeMet from selenohomocysteine is catalyzed by enzyme cobalaminindependent methionine synthase (AB282_01662), where selenohomocysteine undergoes a methylation process to create SeMet. There is a dependence on cobalamin in the activation of methyltransferases, as in that of MetH isolated from E. coli (Thomas and Surdin-Kerjan 1997). Still, both in S. cerevisiae and S. boulardii, homocysteine methyltransferase is independent of cobalamin. This is veri ed since none require vitamin B12 as growth factor.
Additionally, SeMet creates S-adenosyl-selenomethionine through enzyme S-adenosylmethionine synthetase (AB282_03468/AB282_00999), the catalyzer when the adenosyl group of ATP is transferred to the selenium atom of methionine. There, selenohomocysteine is created again by enzyme S-adenosyl-Lhomocysteine hydrolase encoded by AB282_01610, catabolizing S-adenosyl-L-homocysteine formed after the donation of the activated methyl group of S-adenosyl-L-methionine to a receptor. The substitution of methionine by SeMet in proteins does not signi cantly alter the kinetic properties of the enzymes (Kitajima and Chiba 2013).
On the other hand, the biosynthesis of SeCys from selenohomocysteine starts with the conversion of selenohomocysteine into selenocystathionine through the reaction catalyzed by the enzyme cystathionine β-synthase encoded by AB282_01996. The reaction is reversible by the action of the enzyme peroxisomal cystathionine β-lyase (AB282_02293) converting selenocystathionine into selenohomocysteine. A later step is the transformation of selenocystathionine into SeCys by the enzyme cystathionine γ-lyase (AB282_00053). In addition, SeCys is transformed into γ-glutamyl-selenocysteine, the rst step in the biosynthesis of selenoglutathione. Finally, selenoglutathione is formed by the action of glutathione synthetase (AB282_04624), which catalyzes the synthesis of ATP-dependent selenoglutathione from γ-glutamyl-selenocysteine and glycine (Fig. 2).

Conclusions
Due to the identi cation of orthologous genes between S. cerevisiae and S. boulardii, we established the biochemical pathway this probiotic yeast follows for the biotransformation of inorganic selenium into selenomethionine and selenocysteine. In silico studies allow for a theoretical approach of the biochemical mechanisms of a yeast like S. boulardii, which are greatly important to the technological use this yeast offers. The addition of S. boulardii to the processing of fermented foods has advantages, besides the tested probiotic capability, given the relevance of the study of organic selenium as a highly bioaccessible and bioavailable metabolite in the human body, as compared against inorganic selenium usually consumed. The bioaccumulation of selenium by this yeast could create seleno nanoparticles, whose use opens a eld of opportunities and challenges in medicine as alternative therapies against diseases like cancer.

Declartions
Founding Authors declare that this work did not have any funding

Con icts of interest/Competing interests
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