Novel Cysteine-Centered Sulfur Metabolic Pathway in the Thermotolerant Methylotrophic Yeast Hansenula polymorpha

In yeast and filamentous fungi, sulfide can be condensed either with O-acetylhomoserine to generate homocysteine, the precursor of methionine, or with O-acetylserine to directly generate cysteine. The resulting homocysteine and cysteine can be interconverted through transsulfuration pathway. Here, we systematically analyzed the sulfur metabolic pathway of the thermotolerant methylotrophic yeast Hansenula polymorpha, which has attracted much attention as an industrial yeast strain for various biotechnological applications. Quite interestingly, the detailed sulfur metabolic pathway of H. polymorpha, which was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments, revealed the absence of de novo synthesis of homocysteine from inorganic sulfur in this yeast. Thus, the direct biosynthesis of cysteine from sulfide is the only pathway of synthesizing sulfur amino acids from inorganic sulfur in H. polymorpha, despite the presence of both directions of transsulfuration pathway Moreover, only cysteine, but no other sulfur amino acid, was able to repress the expression of a subset of sulfur genes, suggesting its central and exclusive role in the control of H. polymorpha sulfur metabolism. 35S-Cys was more efficiently incorporated into intracellular sulfur compounds such as glutathione than 35S-Met in H. polymorpha, further supporting the cysteine-centered sulfur pathway. This is the first report on the novel features of H. polymorpha sulfur metabolic pathway, which are noticeably distinct from those of other yeast and filamentous fungal species.


Introduction
Sulfur plays important roles in a number of cellular processes, such as the redox cycle (thioredoxins, glutaredoxins), stress response (glutathione, phytochelatins), enzyme reactions (ironsulfur cluster as prosthetic group), and metabolism of secondary products (glucosinolates, sulfated compounds) [1,2]. It is also essential in C1 metabolism as a source of reduced sulfur for the biosynthesis of S-adenosyl-methionine (AdoMet). Cellular requirements for sulfur can be fulfilled by the uptake of sulfur-containing amino acids, cysteine and methionine, or by the assimilation of inorganic sulfur into organic compounds such as cysteine and homocysteine, which are used for further biosynthesis of glutathione (GSH) and methionine, respectively [3]. Different from microorganisms and plants, animals do not have the assimilatory mechanisms for inorganic sulfur, and they require methionine as an essential amino acid for their source of sulfur nutrient. In the yeast and filamentous fungal species, cysteine biosynthesis from sulfide can be divided into two pathways [4,5]. In one pathway, sulfide is condensed with O-acetylhomoserine to generate homocysteine, which can be converted to cystathionine and then to cysteine, as in Saccharomyces cerevisiae (Fig.1A). In the other pathway, sulfide is condensed with O-acetylserine to generate cysteine in a process catalyzed by cysteine synthase (OAS pathway), as in Schizosaccharomyces pombe (Fig.1B). The filamentous fungi Aspergillus nidulans and Neurospora crassa employ both pathways for cysteine biosynthesis (Fig. 1C). In S. cerevisiae, A. nidulans, and N. crassa, cysteine and homocysteine interconvert through forward and reverse transsulfuration pathways. In contrast, S. pombe lacks the reverse pathway for conversion of homocysteine to cysteine due to lack of required enzymes cystathionine b-synthase and cystathionine c-lyase [6,7].
The thermotolerant methylotrophic yeast Hansenula polymorpha is characterized by its high tolerance to various stresses induced by heavy metals, xenobiotics (drugs), and environmental pollutants. As a consequence it has attracted much attention as a promising host strain for recombinant protein production [8] but also as an industrial yeast strain for various biotechnological applications [9]. In basic research, it has long been used as a favorable model system to study peroxisome biogenesis and function due to extensive peroxisome proliferation during growth on methanol [10]. Since methylotrophic yeasts are obligatorily dependent on thiol GSH for oxidation and detoxification of formaldehyde, a toxic methanol oxidation intermediate [11], H. polymorpha is considered as a good model system to study the metabolism and functions of GSH [12,13] and a promising host strain for high level production of GSH [14]. Even though the sulfur metabolism is important for methylotrophic growth of H. polymorpha, its metabolic pathways involved in the biosynthesis of GSH and other sulfur compounds is not well understood.
In this study, a detailed sulfur metabolic pathway of H. polymorpha was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments. In addition, we examined the effect of sulfurcontaining amino acids on the transcriptional regulation of H. polymorpha sulfur pathway. Here, we showed the novel features of the sulfur metabolic pathway and its regulation in H. polymorpha, which are notably distinct from those of other yeast and filamentous fungal species.

Strains, media and cultivation conditions
The H. polymorph DL1-LdU (leu2 ura3::lacZ ) strain was used as a parental strain and the mutant H. polymorpha strains constructed in this study are listed in Table 1. The null mutant strain of ScGSH1 was constructed by replacing the coding region of ScGSH1 with the ScLEU2 gene in S. cerevisiae L3262 (MATa ura3-52 leu2-3, 112 his4-34). Yeast cells were cultivated in YPD (1% yeast extract, 2% peptone, and 2% glucose), YNB (0.67% yeast nitrogen base without amino acids and 2% glucose) or sulfur-free B-medium (synthetic medium with 2% glucose without any sulfur source) [4]. Inorganic (sulfate, sulfite, and sulfide) and organic (methionine, cysteine, cystathionine, homocysteine, and GSH) sulfur sources were added to B-medium at 2 mM and 0.2 mM final concentration, respectively. For solid B-medium, 1% agarose was used instead of agar to minimize the addition of uncontrolled sulfur sources to the medium. Uracil (20 mg/ml), leucine (100 mg/ml), and histidine (20 mg/ml) were added to the growth media according to the auxotrophic requirements of individual strains. For heavy metal cadmium exposure, yeast cells were cultured in YPD containing cadmium sulfate (C2919, Sigma-Aldrich, St. Louis, MO, USA).

Construction of mutant strains deficient in OAS pathway and transsulfuration pathway
The complete DNA sequences of genes involved in cysteine and methionine biosynthesis were obtained from the in-house database of H. polymorpha DL1-L (Korea Research Institute of Bioscience and Biotechnology, Korea). Disruption of these genes was carried out by the modified fusion PCR-based gene deletion method as described previously [15] using gene-specific primers (Table S1). The H. polymorpha DL1-LdU (leu2 ura3) strain was transformed with PCR product carrying the gene disruption cassette with the HpURA3 pop-out marker and transformants were selected on SC-URA medium supplemented with 0.1 mM GSH, methionine, or cysteine. Correct replacement of the target gene was confirmed by PCR analysis using primers NF and CR. To generate multiple gene disruptions, ura3 revertants of a disruption mutant were selected on YPD agar plates containing 5-fluoroorotic acid (5-FOA; 0.5 mg/ml) and subjected to the next round of the fusion PCR-based gene deletion method.

Quantitative reverse transcription PCR analysis
For quantitative reverse transcription PCR (qRT-PCR) analysis, cDNA was synthesized from total RNA using Superscript reverse transcriptase (Invitrogen, Carlsbad, CA). qRT-PCR was performed in Rotor-Gene Q (Qiagen) with a QuantiMix SYBR Kit (Philekorea Technology, Daejeon, Korea) using gene-specific primer sets (Table S1). Each sample was analyzed in duplicate and normalized to b-actin as an endogenous control. The relative concentrations of mRNAs were calculated using 2 2DDCt methodology.

Analysis of GSH synthesis by 35 S labeling
Yeast cells were cultivated in YNB medium supplemented with uracil, leucine, and histidine at 30uC for S. cerevisiae and 37uC for H. polymorpha. When the yeast culture reached mid-log phase (A 600 = 0.3), 2-ml aliquots were withdrawn and incubated with [ 35 S]methionine (200 mCi) or [ 35 S]cysteine (200 mCi) for 2 hr in the presence of 0, 0.6, or 2 mM CdSO 4 . Cells were collected by centrifugation, washed with water, and resuspended in 50 ml of water. Cells were then boiled for 5 min and centrifuged to obtain the 35 S-labeled metabolites in the supernatant. The metabolites were oxidized by addition of an equal volume of performic acid [16], which converts both oxidized and reduced forms of glutathione to glutathione-sulfonic acid. Samples (5 ml) were applied onto cellulose thin layer chromatography in the following solvent system: butanol-1/acetic acid/water (90:15:33) [17]. The 35 S-labeled metabolites on the thin layer chromatography plate were scanned and quantified by phosphor technology (BAS-1500, FUSIFILM, Japan).

Bioinformatic analysis
Multiple sequence alignments were constructed with CLUS-TALW method of the DNASTAR MegAlign program and shaded using GeneDoc (http://www.nrbsc.org). Percentage identity was calculated by comparing sequence pairs in relation to the phylogeny reconstructed by the CLUSTALW method of the DNASTAR MegAlign program [18].

Results
In silico reconstruction of the sulfur metabolic pathway in H. polymorpha Based on in-house genome information on the H. polymorpha DL-1 strain (KRIBB in Korea), we have reconstructed the putative sulfur metabolism pathway involved in sulfate assimilation, sulfur amino acid biosynthesis, and the methionine salvage pathway ( Fig. 2). The nucleotide sequences of H. polymorpha DL-1 genes involved in sulfur metabolic pathway were deposited in GenBank under accession numbers JN676924-JN676946 (Table S2). For sulfur incorporation into carbon chains, we identified a H. polymorpha gene predicted to participate in the direct biosynthesis of cysteine from inorganic sulfur compounds, namely the cysteine synthase gene, which is absent in S. cerevisiae and Candida glabrata [19]. Using the A. nidulans cysB and S. pombe CYS1a genes encoding cysteine synthase as queries for the search, we identified a candidate ORF designated HpCYS1 whose deduced amino acid sequence shows 59% and 54% identity to those of A. nidulans cysB and S. pombe CYS1a proteins, respectively (Fig. 3A). The putative HpCYS1 protein (HpCys1p) has two lysine residues that can bind the pyridoxal 59-phosphate cofactor and are highly conserved among cysteine synthases of plants and bacteria. The presence of a functional OAS pathway in H. polymorpha was further suggested by the identification of a H. polymorpha homolog (designated HpSAT1) of A. nidulans cysA encoding serine O-acetyltransferase [20], which could provide a substrate of HpCys1p. The putative HpSAT1 protein (HpSat1p) showed high identity to A. nidulans CysAp (59.0%) compared to low identities to ScMet2p (24.1%) and HpMet2p (27.3%) encoding homoserine O-acetyltransferase (Fig. 3B).
Interestingly, analysis of the H. polymorpha genome indicates that H. polymorpha does not have an ORF coding for O-acetylhomoserine sulfhydrylase or homocysteine synthase (encoded by MET17 or MET25 in S. cerevisiae), which catalyzes the synthesis of homocysteine from homoserine by incorporation of sulfide. In fact, the absence of a MET17 homolog in H. poymorpha was implicated in a previous study reporting the methionine auxotrophic phenotype of a H. poymorpha mutant strain with a defect in a gene coding for a homolog of S. cerevisiae Str3p [21]. Considering that homocysteine is the central molecule for the biosynthesis of sulfur amino acids in all organisms studied [19], the absence of de novo synthesis of homocysteine from inorganic sulfur might be an exceptional feature of H. polymorpha. We also searched for other H. polymorpha genes showing significant identities to S. cerevisiae genes involved in the transsulfuration pathway, including HpSTR2 (cystathionine c-synthase) and HpSTR3 (cystathionine b-lyase) for forward transsulfuration, and HpCYS3 (cystathionine c-lyase) for reverse transsulfuration. Three H. polymorpha ORFs with quite low identity to ScCYS4 (cystathionine b-synthase) were identified and designated HpCYS4a, HpCYS4b, and HpCYS4c. For the methyl cycle of methionine and AdoMet biosynthesis, only one copy of a SAM2 homolog was identified in the H. polymorpha genome, whereas two copies of genes encoding S-adenosylmethionine synthetase are present in S. cerevisiae (Table S2).

Validation of H. polymorpha sulfur pathway by systematic deletion analysis
To determine whether homologs identified by in silico analysis play physiological roles in sulfur metabolism, a set of H. polymorpha mutants were constructed by gene deletion (Table 1). Phenotypic analysis of H. polymorpha mutant strains was performed on B-media supplied with sulfate, cystathionine, cysteine, or methionine as the only sulfur source. Since H. polymorpha does not possess a MET17 homolog, the reaction mediated by HpCys1p should be the only way to synthesize the sulfur amino acid de novo from inorganic sulfur. As predicted by the absence of MET17 in H. polymorpha, the Hpcys1D mutant strain was not able to grow on B medium supplemented with sulfate as the only sulfur source (Fig. 4A). Growth of Hpcys1D on B-medium was recovered by supplementation with any of cystathionine, cysteine, or methionine as a sole sulfur source (Fig. 4B), strongly indicating that cysteine and methionine can be interconverted in H. polymorpha through the transsulfuration pathway in both directions with cystathionine as an intermediate.
The transsulfuration reactions involve at least four enzymes, Str2p, Str3p, Cys3p, and Cys4p [5,7,22,23]. To more systematically validate the presence of an active transsulfuration pathway in both directions with allocation of functional genes in H. polymorpha, a set of double disruption mutants (Hpcys1Dcys3D, Hpcys1Dstr2D,  Hpcys1Dstr3D, Hpcys1Dcys4aD, and Hpcys1Dcys4bD) and a quadru-  ple disruptant (Hpcys1Dcys4aDcys4bDcys4cD) were constructed in the background of the Hpcys1D strain. Although all deletion mutants were viable and morphologically normal in YPD medium, they displayed growth defects to different degrees on various sulfur sources (Fig. 4B). Growth of the HpSTR disruption mutants, such as Hpcys1Dstr2D and Hpcys1Dstr3D, was possible only with supplementation of methionine, but not cysteine, supporting the notion that HpStr2p and HpStr3p are responsible for the forward transsulfuration reaction from cysteine to methionine in H. polymorpha. On the other hand, Hpcys1Dcys3D could only grow on cysteine, but not on cystathionine or methionine (Fig. 4B), strongly supporting the essential role of HpCys3p in the reverse transsulfuration in H. polymorpha.
Unexpectedly, none of the single deletion strains of HpCYS4 homologs showed any apparent growth defect phenotype. Even the quadruple Hpcys1Dcys4aDcys4bDcys4cD null mutant was viable and morphologically normal in methionine-supplemented minimal medium (Fig. 4B), indicating that other proteins can perform the function of cystathionine b-synthase.
The presence of an active OAS pathway in H. polymorpha was further supported by analysis of the growth phenotypes of the HpSAT1 disruptant (Hpsat1D), which were clearly distinctive from those of the HpMET2 disruptant (Hpmet2D) with deletion of the HpMET2 gene encoding homoserine O-acetyltransferase [24]. While growth of Hpsat1D on B-medium can be supported by supplementation with cysteine or methionine, growth of Hpmet2D is strictly dependent on the addition of methionine (Fig. 4C). The similar growth patterns of Hpsat1D and Hpcys1D, as shown in Fig. 4, strongly suggest that the role of HpSat1p is to generate a substrate for the reaction mediated by HpCys1p. In particular, the Hpcys1D and Hpsat1D strains showed an apparently reduced growth phenotype compared with the wild type strain even on YPD medium, indicating the importance of the OAS pathway in the generation of cysteine, which serves as the base for the biosynthesis of sulfur amino acids in H. polymorpha.
In methylotrophic yeasts, antioxidant activity is particularly essential for methanol metabolism, which produces hydrogen peroxide [25]. Considering that cysteine is the rate-limiting nutrient in the biosynthesis of GSH, an essential antioxidant molecule involved in oxidative stress response and detoxification for methylotrophic growth, it is plausible that H. polymorpha might have evolved to have a predominantly cysteine-centered sulfur pathway to maximize GSH biosynthesis. It is noteworthy that the Hpsat1D strain displayed increased sensitivity not only to oxidative stress and high temperature but also to Cd (Fig. 5), indicating that de novo cysteine biosynthesis from inorganic sulfur via the OAS pathway is an essential step in providing sulfur compounds required for the inherent resistance of H. polymorpha to Cd, oxidative, and heat stresses.

Regulation of the sulfur metabolic pathway in H. polymorpha by sulfur amino acids
To obtain information on transcriptional regulation of the sulfur pathway, we performed preliminary northern blot analysis on a few selected genes, such as HpMET3, HpMET10, HpCYS1, and HpCYS3 involved in the sulfur pathway and analyzed the effect of sulfur limitation and Cd exposure on their expression ( Figure S1). It was shown that although the transcripts of HpMET3, HpMET10, and HpCYS3 were barely detectable in YPD medium, they were highly expressed in medium B, indicating the induced expression under sulfur limitation condition ( Figure S1A, left panel). Interestingly, the basal level of the HpCYS1 transcript was detected significantly high even on YPD medium. Upon exposure to Cd, the induced expression of HpMET3, HpMET10, and HpCYS3 was also observed ( Figure S1A, right panel). It is notable that the induction of HpMET3, HpMET10, and HpCYS3 by sulfur limitation could be repressed significantly by addition of 1 mM cysteine to B-medium, but was not greatly repressed by 1 mM methionine ( Figure S1B). Moreover, supplementation with methionine rather strongly induced transcription of HpCYS1. Our northern blot data led to speculation that the expression of sulfur metabolic pathway genes in H. polymorpha is tightly subject to down-regulation by cysteine, but not by methionine supplementation.
In S. cerevisiae, the repression effect of methionine on the expression of sulfur metabolic pathway genes is attributed to its conversion to cysteine [26]. To investigate the possibility that the marginal repressive effect of methionine on the expression of sulfur metabolic pathway genes is due to the intrinsic low uptake rate of methionine by H. polymorpha, we examined the repressive effect of supplementation with AdoMet, which would be converted to homocysteine through the methyl cycle and then transformed into cysteine via the reverse transsulfuration pathway (Fig. 6). The quantitative reverse transcription PCR (qRT-PCR) analysis of HpMET2, HpSAT1, HpSTR2, HpSTR3, HpGSH2, and HpCYS3 were carried out, together with HpSUL1, HpMET3, and HpMET10. The RNA samples were obtained from cells cultivated in YPD, B-medium, and B-medium supplemented with 0.5 mM methionine, cysteine, or AdoMet. It was observed that all the tested genes were induced in B-medium as indicated in northern blot analysis. Quite unexpectedly, we found that expression of  HpMET2, HpSTR3, and HpCYS3, which are involved in the transsulfuration pathway, was not repressed even by the addition of AdoMet, while the expression of HpMET3, HpSUL1, and HpSAT1, which are involved in sulfur assimilation, was efficiently repressed by the addition of AdoMet (Fig. 6). Semi-quantitative PCR analysis also confirmed the differential effect on transcriptional repression of H. polymorpha sulfur genes by methionine, cysteine, and AdoMet ( Figure S2). This is an unexpected feature of H. polymorpha sulfur metabolism, considering that methionine, AdoMet, and cysteine repress MET gene expression equally efficiently in S. cerevisiae [26]. We also analyzed the expression pattern of HpGSH1 and HpGSH2, encoding key enzymes responsible for the synthesis of GSH and observed that their expressions were also repressed only by the supplementation of cysteine, not by Met and AdoMet.

Analysis of glutathione biosynthesis in H. polymorpha
Given the importance of sulfur metabolic pathway in the biosynthesis of GSH, which is an essential sulfur compound for detoxification of Cd, we investigate GSH biosynthesis in H. polymorpha with comparison to that of S. cerevisiae under Cd stress conditions (Fig. 7). After 2 hr incubation with [ 35 S]methionine ( 35 S-Met) or [ 35 S]cysteine ( 35 S-Cys) in the presence of 0, 0.6, or 2 mM CdSO 4 , cells were harvested and the extracted metabolites were separated by thin layer chromatography (TLC). Upon Cd treatment, the synthesis level of 35 S-GSH increased more than 4fold in the S. cerevisiae wild-type strain labeled with either 35 S-Cys (Fig. 7A, left panel) or 35 S-Met (Fig. 7B, left panel). In contrast, the 35 S-GSH biosynthesis from 35 S-Cys or 35 S-Met was significantly decreased in Scgsh1D, as expected. It is interesting to note that the level of 35 S-cystathionine, an intermediate metabolite in the transsulfuration pathway also increased with an increase in Cd concentration in both labeling reactions. This is consistent with a previous report of an increased flux toward the transsulfuration branch followed by GSH synthesis in S. cerevisiae upon exposure to Cd [27]. The residual GSH biosynthesis activity in the Scgsh1D mutants could be explained by the presence of another minor system for GSH biosynthesis through the Pro1 and Pro2 enzymes involved in proline biosynthesis in S. cerevisiae [28].
In the case of H. polymorpha, it is quite noteworthy that 35 S-GSH was detected after 2 hr incubation with 35 S-Cys (Fig. 7A, right panel) but hardly with 35 S-Met (Fig. 7B, right panel). Even with 35 S-Cys, 35 S-labeled cystathionine was not detected in TLC with the samples from H. polymorpha. Moreover, the increase in GSH biosynthesis upon Cd exposure was not as obvious in H. polymorpha as in S. cerevisiae, although we noticed increased accumulation of an unidentified 35 S-labeled intermediate, designated X, in a Cd concentration-dependent manner in H. polymorpha. In addition, analysis of the total amount of intracellular 35 S-labeled metabolites showed apparently higher incorporation efficiency of 35 S-Cys than that of 35 S-Met in H. polymorpha ( Figure S3), suggesting intrinsic low uptake capacity of H. polymorpha for methionine.

Discussion
The comparative genomic study of sulfur metabolism of hemiascomycetous yeasts [19] indicated that homocysteine is the base for the biosynthesis of sulfur amino acids in the traditional yeast S. cerevisiae and most hemiascomycetous yeasts (Fig. 1A). In S. cerevisiae, sulfide is incorporated into a three-carbon chain through formation of homocysteine, followed by synthesis of methionine and cysteine from homocysteine through either the methyl cycle (leading to the synthesis of methionine and AdoMet) or the reverse transsulfuration pathway (leading to synthesis of cysteine and glutathione) [29]. The filamentous fungi, such as A. nidulans and N. crassa, and the fission yeast have an additional mechanism for sulfide incorporation into carbon chains to give cysteine through the OAS pathway ( Fig. 1B and 1C). Moreover, interconversion of sulfur amino acids (methionine-homocysteine-cysteine) occurs through both forward and reverse transsulfuration pathways in the filamentous fungi, which have been reported to have the richest repertoire of sulfur metabolic options. In contrast, S. pombe has only one pathway with forward transsulfuration. It was suggested that wild type S. pombe can utilize methionine as a sulfur source only after it is degraded to give rise to sulfate [6]. Similar to S. cerevisiae and filamentous fungal species, our data strongly indicated that H. polymorpha also possesses both forward and reverse transsulfuration pathways (Fig. 1D). Thus, like S. cerevisiae, H. polymorpha can grow in the presence of either methionine or cysteine as the sole sulfur source. However, a novel feature of H. polymorpha is the lack of direct incorporation of sulfide into homocysteine due to the absence of a MET17 (or MET25) homolog. As a result, biosynthesis of methionine in H. polymorpha is possible only through cysteine and cystathionine, i.e. the forward transsulfuration reaction, which is mediated by HpSTR2 and HpSTR3 proteins. It is quite intriguing that sulfur metabolism of H. polymorpha is centered on cysteine rather than methionine, in contrast to other hemiascomycetes such as S. cerevisiae and Kluyveromyces lactis [30]. The comparative analysis of 35 S-Met and 35 S-Cys labeling experiments (Fig. 7, Figure S3) showed that the incorporation efficiency of cysteine into sulfur compounds was much higher than that of methionine in H. polymorpha, which is quite different from S. cerevisiae. The use of cysteine as preferential organic sulfur source partly reflects the cysteine-centered sulfur metabolism of H. polymorpha. Moreover, genome sequence analysis reveals that H. polymorpha possesses HpYCT1, a homolog of the S. cerevisiae ScYCT1 encoding a high-affinity cysteine-specific transporter [31], but no homolog of the S. cerevisiae MUP1 encoding a high-affinity methionine permease [32]. Furthermore, in contrast to S. cerevisiae, which possesses a family of multiple methionine transporter genes, H. polymorpha has only one ORF encoding methionine transporter, HpMUP3, which is a homolog of S. cerevisiae MUP3 encoding a lowaffinity methionine permease.
Our qRT-PCR data on transcript level analysis (Fig. 6) strongly suggest that the entire sulfur assimilation pathway leading to cysteine biosynthesis has evolved to be up-regulated upon sulfur limitation in H. polymorpha. Considering cysteine-centered sulfur metabolic pathway of H. polymorpha is quite unique among those of other yeast and filamentous fungal species, it will be intriguing to investigate the regulatory mechanisms of H. polymorpha sulfur pathway. Especially, the role of a H. polymorpha homolog of S. cerevisiae Met4p, a positive trans-acting sulfur regulatory protein related to the bZIP protein family [23], would be an intriguing subject for further study. While carrying out sequence analysis, we noticed that H. polymorpha Met4p is considerably smaller (330 amino acid residues) than ScMet4p (672 amino acid residues), reflecting different structural organization between two yeast Met4 homologs (Table S2). Therefore, molecular genetics and functional genomic studies on regulatory network mediated by HpMet4p would provide further insight into how this novel sulfur metabolic pathway is regulated in H. polymorpha, broadening our basic understanding of the conservation and divergence of the sulfur metabolic networks among eukaryotic organisms. On the other hand, from a biotechnological viewpoint, comprehensive knowledge of the sulfur pathway and its regulation might be usefully applied in the development of the artificial genetic circuit by finetuning of sulfur metabolism in yeast and filamentous fungal species to produce high-valued sulfur-containing amino acid and metabolites. Figure S1 Northern blot analysis of transcriptional regulation of HpMET3, HpMET10, HpCYS1, and HpCYS3. Yeast cells were grown in YPD to the exponential phase and then transferred to B-medium. For Cd exposure, yeast cells grown in YPD to the exponential phase were transferred to YPD medium containing 0.6 mM Cd. After 2 hr cultivation, yeast cells were harvested and total RNA was extracted using the hotphenol method. Total RNA was electrophoresed on 1.2% agaroseformaldehyde gels, blotted overnight onto a Nylon + membrane, and hybridized with 32 P-labeled DNA probes. DNA probes were labeled with the Rediprime II random priming labeling system kit (GE healthcare). (TIF) Figure S2 Semi-quantitative reverse transcriptase-PCR (semiRT-PCR) analysis of transcriptional regulation of H. polymorpha genes in the sulfur pathway. Expression of a selected set of genes was analyzed by semiRT-PCR. Yeast cells were grown in YPD to the exponential phase and then transferred to B-medium supplemented with the indicated 0.5 mM sulfur compounds. After 2 hr cultivation, yeast cells were harvested and total RNA was extracted for analysis.