Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

doi:10.21203/rs.3.rs-762539/v1

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Research Article

Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

https://doi.org/10.21203/rs.3.rs-762539/v1

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published 16 Oct, 2021

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Background

In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis. Here, we investigate DHODs from three fungi (the Neocallimastigomycete Anaeromyces robustus and the yeasts Schizosaccharomyces japonicus and Dekkera bruxellensis) that can grow anaerobically but, based on genome analysis, only harbor a Class-II DHOD.

Results

Heterologous expression of putative Class-II DHOD-encoding genes from fungi capable of anaerobic, pyrimidine-prototrophic growth (Arura9, SjURA9, DbURA9) in an S. cerevisiae ura1Δ strain supported aerobic as well as anaerobic pyrimidine prototrophy. GFP-tagged SjUra9 and DbUra9 were localized to S. cerevisiae mitochondria, while ArUra9, whose sequence lacked a mitochondrial targeting sequence, was localized to the yeast cytosol. Experiments with cell extracts showed that ArUra9 used free FAD and FMN as electron acceptors. A strain expressing DbURA9 initially grew slowly without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. Expression of SjaUra9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA, which coincided with the natural respiratory deficiency of Sch. japonicus. A cysteine residue (C265 in SjUra9) in the active sites of all three anaerobically active Ura9 orthologs was shown to be essential for anaerobic activity of SjUra9 but not of ArUra9.

Conclusions

Activity of fungal Class-II DHODs was long thought to be dependent on an active respiratory chain, which in most fungi requires the presence of oxygen. By heterologous expression experiments in S. cerevisiae, this study shows that phylogenetically distant fungi independently evolved Class-II dihydroorotate dehydrogenases that enable anaerobic pyrimidine biosynthesis. Further structure-function studies are required to understand the mechanistic basis for the anaerobic activity of Class-II DHODs and an observed loss of respiratory competence in S. cerevisiae strains expressing an anaerobically active DHOD from Sch. japonicus.

Biotechnology and Bioengineering

Yeast

Neocallimastigomycota

oxygen

anaerobiosis

oxygen requirements

uracil

Oxidation of dihydroorotate to orotate, an essential reaction for pyrimidine biosynthesis in all domains of life, is catalyzed by the flavoprotein dihydroorotate dehydrogenase (DHOD) [1, 2]. Depending on the type of DHOD, different electron acceptors are used for reoxidation of the flavin cofactor. Soluble Class-I DHODs include homodimeric fumarate-dependent type I-A enzymes and heterotetrameric NAD⁺-dependent type I-B enzymes [3, 4], while membrane-bound Class-II DHODs are quinone-dependent and coupled to respiration [5]. Protein sequence identity between Class-I and Class-II DHODs is only approximately 20% [6–8].

Class I-A and Class-II DHODs contain a single FMN cofactor per subunit. In heterotetrameric Class I-B enzymes, two subunits also each contain an FMN cofactor, while the other two both contain an FAD cofactor and an [2Fe-2S] cluster [4]. Class-I DHODs contain an active-site cysteine residue involved in deprotonation of C5 of dihydroorotate, while Class-II enzymes have a serine in the same position acting as the catalytic base [9–13].

Most eukaryotes harbor a monomeric Class-II DHOD that donates electrons to the quinone pool of the mitochondrial respiratory chain [8, 14–16]. Bacterial Class-II DHODs have an N-terminal sequence that localizes them to the inside of the cytoplasmic membrane, whereas eukaryotic enzymes are targeted to the outside of the mitochondrial inner membrane [5]. This mitochondrial targeting also applies to fungal Class-II DHODs, which include the Ura9 orthologs of yeasts such as Lachancea kluyveri and Schizosaccharomyces pombe [17]. Since respiration in yeasts requires oxygen as electron acceptor, reliance on these respiration-coupled enzymes precludes pyrimidine-prototrophic anaerobic growth [8].

Class-I DHODs predominantly occur in gram-positive bacteria and Archaea [18] but are also found in a small number of yeasts, including Saccharomyces cerevisiae and closely related species [19, 20]. S. cerevisiae is among the few yeast species that are able to grow under strictly anaerobic conditions [21]. ScUra1, a Class-IA, fumarate-coupled DHOD, enables S. cerevisiae to synthesize pyrimidines in the absence of oxygen [19, 20]. A small number of other Saccharomycetes, including Kluyveromyces lactis and Lachancea kluyveri, harbor Ura1 as well as Ura9 orthologs [19, 22, 23]. Based on sequence similarity of yeast ScURA1 orthologs with Lactococcus genes, they are assumed to have been acquired by horizontal gene transfer [15, 19].

In line with a proposed essentiality of ScURA1 orthologs for anaerobic pyrimidine synthesis by yeasts [19, 24], replacement of ScURA1 by a Class-II DHOD gene from L. kluyveri (LkURA9) or Schizosaccharomyces pombe (SpURA3) yielded strains that were only pyrimidine prototrophic under aerobic conditions [8, 19]. Conversely, replacement of ScURA1 by LkURA1 supported aerobic as well as anaerobic pyrimidine prototrophy [19]. Introduction of ScURA1 in URA9-dependent yeasts was proposed as a metabolic engineering strategy for enabling anaerobic, pyrimidine-prototrophic growth of yeasts lacking a native ScURA1 ortholog [25].

The long-held assumption that expression of a Class-I DHOD is required for anaerobic pyrimidine biosynthesis in eukaryotes was first challenged when D. bruxellensis, which only harbors a URA9 ortholog, was shown to grow anaerobically in pyrimidine-free media [26]. A hypothesis that DbUra9 is able to use a non-quinone electron acceptor [17, 27] was, however, not experimentally tested.

We recently observed that D. bruxellensis may not be the only eukaryote in which anaerobic pyrimidine synthesis involves a Class-II DHOD. Inspection of the genome of the fission yeast Schizosaccharomyces japonicus, which shows fast anaerobic growth in synthetic media without uracil [28, 29], suggested that it only contains a URA9 ortholog. Moreover, genomes of Neocallimastigomycetes, a group of deep-branching, obligately anaerobic fungi that lack mitochondria and instead harbor hydrogenosomes [30, 31], also appeared to lack orthologs of soluble Class-I DHOD.

The goals of the present study were to investigate whether URA9 orthologs in eukaryotes capable of anaerobic growth indeed support anaerobic pyrimidine biosynthesis, and to gain more insight into underlying mechanisms and trade-offs. To this end, we expressed putative Class-II DHOD genes from the obligately anaerobic Neocallimastigomycete Anaeromyces robustus (Arura9), the facultative anaerobes Sch. japonicus (SjURA9) and D. bruxellensis (DbURA9), as well as from the oxygen-requiring yeasts O. parapolymorpha (OpURA9) and K. marxianus (KmURA9), in an

S. cerevisiae ura1Δ background. After studying aerobic and anaerobic growth of the resulting strains in uracil-supplemented and uracil-free synthetic media, we analyzed subcellular localization of Ura9-eGFP fusion proteins in S. cerevisiae and assessed the impact of a conserved amino-acid substitution in anaerobically functional Ura9 orthologs. To identify possible natural electron acceptors, we performed enzyme assays in cell extracts of an S. cerevisiae strain expressing ArUra9 and re-sequenced the genomes of laboratory-evolved S. cerevisiae strains whose anaerobic growth depended on expression of DbURA9. We found that instead of quinone, ArUra9 uses free flavins as electron acceptors and that expression of SjURA9 in S. cerevisiae results in loss of respiration.

Obligately anaerobic Neocallimastigomycetes and facultatively anaerobic yeasts harbor putative Class-II DHODs. A preliminary exploration of the occurrence of Class-I and Class-II DHODs in selected fungal proteomes was based on sequence comparisons with the Class-I and Class-II enzymes of Lanchancea kluyveri (LkUra1 and LkUra9, respectively; [19]) (Table 1). Consistent with earlier studies, the Saccharomyces cerevisiae proteome only showed a sequence with strong similarity to LkUra1 (ScUra1; [10, 19, 23]). Similarly, proteomes of the yeasts Ogataea parapolymorpha and Kluyveromyces marxianus, which both require oxygen for growth [32, 33] yielded previously described Class-II DHOD sequences with strong homology to LkUra9 (OpUra9 and KmUra9, respectively; [23, 34]). As previously described, K. marxianus also showed a sequence with high sequence similarity to LkUra1 (KmUra1; [22]) while the facultatively anaerobic yeast Dekkera bruxellensis only showed a sequence with high similarity to LkUra9 (DbUra9; [17, 23, 27]). Sequence comparison with LkUra1 provided no indication for the presence of a Class-I DHOD in the obligately anaerobic Neocallimastigomycetes Piromyces finnis, Neocallimastix californiae and Anaeromyces robustus. Instead, single predicted protein sequences with high similarity to the Class-II DHOD LkUra9 were identified in these species (Table 1) and tentatively called PfUra9, NcUra9 and ArUra9, respectively. A similar result was obtained for the facultatively anaerobic fission yeast Schizosaccharomyces japonicus [28, 29], whose putative Class-II DHOD was tentatively named SjUra9 (Table 1).

Table 1

Lanchancea kluyveri **LkUra1 and LkUra9 sequence homology results using selected fungal proteomes.** Proteomes of the Neocallimastigomycetes *A. robustus* (NCBI taxid 1754192), *P. finnis* (1754191), *N. californiae* (1754190), and the yeasts *Sch. japonicus* (402676), *D. bruxellensis* (5007), *K. marxianus* (1003335), *O. parapolymorpha* (871575) and *S. cerevisiae* (559292) were subjected to blastp searches using LkUra1 (DHOD Class I-A, UniProt KB accession number Q7Z892) and LkUra9 (DHOD Class II, accession number Q6V3W9) amino acid sequences as queries.
Subject proteome	Resulting GenBank accession	Query coverage (%)		E-value		Identity (%)		Interpretation
Subject proteome		LkUra1	LkUra9	LkUra1	LkUra9	LkUra1	LkUra9	Ura1	Ura9
D. bruxellensis	XP_041139490.1	95	90	5∙10^− 17	4∙10^− 145	24.5	50.9	No	Yes
A. robustus	ORX87218.1	56	77	3∙10^− 9	2∙10^− 85	27.0	44.6	No	Yes
P. finnis	ORX52621.1	56	77	5∙10^− 9	7∙10^− 86	24.9	44.9	No	Yes
N. californiae	ORY72481.1	54	74	2∙10^− 8	5∙10^− 82	27.7	44.9	No	Yes
Sch. japonicus	XP_002171492.1	63	97	2∙10^− 13	1∙10^− 113	30.6	44.3	No	Yes
S. cerevisiae	NP_012706.1	100	71	0.0	4∙10^− 9	80.3	23.8	Yes	No
K. marxianus	XP_022674337.1 XP_022675611.1	100 94	73 95	0.0 6∙10^− 11	2∙10^− 12 0.0	77.1 24.0	25.1 73.7	Yes No	No Yes
O. parapolymorpha	XP_013936870.1	95	91	1∙10^− 9	0.0	23.0	63.4	No	Yes
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To study the phylogeny of fungal Ura9 orthologs, the amino-acid sequence of LkUra9 was used as query for a sequence analysis using a hidden Markov model method (HMMER; [35]) against all fungal proteomes in Uniprot [36], and best hits were used for orthology prediction (see Methods). A similar strategy was performed to obtain all possible bacterial LkUra9 orthologs. The resulting 331 fungal and 73 bacterial Ura9 orthologs were then used to build a phylogenetic tree (Fig. 1, Additional files 1–3). This phylogenetic analysis revealed that LkUra9 orthologs from obligate anaerobic fungi and from the facultative anaerobic yeasts Sch. japonicus and D. bruxellensis do not share a common ancestor. Furthermore, bacterial Ura9 orthologs were clearly separated from fungal Ura9 orthologs. These results indicated that, if Ura9 orthologs of Neocallimastigomycetes, D. bruxellensis and Sch. japonicus have properties that enable anaerobic pyrimidine synthesis, these are likely to have evolved independently, and without involvement of horizontal gene transfer (HGT).

Dependence of anaerobic, pyrimidine-prototrophic growth of phylogenetically distant fungi on a Class-II (‘Ura9’) DHOD is remarkable in view of the reported coupling of eukaryotic Class-II DHODs to mitochondrial aerobic respiration. The origins of these particular Class-II DHODs differ from the proposed acquisition by HGT of respiration-independent Class-I DHODs by an ancestor of S. cerevisiae [19, 20] and from proposed HGT-driven adaptations of Sch. japonicus [29, 37] and Neocallimastigomycetes [38, 39] to anaerobic growth.

Heterologous URA9 genes complement aerobic pyrimidine auxotrophy of ura1Δ

S. cerevisiae. To assess and compare functionality of putative Class-II DHOD genes of the Neocallimastigomycete A. robustus and of the facultatively anaerobic yeasts Sch. japonicus and

D. bruxellensis, the ScURA1 open-reading frame of S. cerevisiae was replaced by expression cassettes for codon-optimized Arura9, SjURA9 or DbURA9 coding sequences (Additional file 4). As a reference, ScURA1 was replaced by expression cassettes for URA9 coding sequences of the aerobic yeasts

K. marxianus and O. parapolymorpha. The S. cerevisiae reference strain CEN.PK113-7D (URA1) showed fast aerobic growth (0.36 h^− 1) on glucose-containing synthetic media with and without uracil (SMUD + ura and SMUD, respectively). As anticipated, the congenic ura1Δ strain IMK824 grew on SMUD + ura (0.30 h^− 1) but not on SMUD (Additional file 5; Table S1). A lower specific growth rate of strain IMK824 on SMUD + ura than observed for CEN.PK113-7D probably reflected a growth-limiting rate of uracil uptake by uracil-auxotrophic S. cerevisiae [40].

S. cerevisiae strains in which expression cassettes for Arura9, DbURA9, KmURA9 or OpURA9 replaced ScURA1, all grew aerobically on SMUD + ura as well as on SMUD, at specific growth rates (0.34 h^− 1 to 0.35 h^− 1) that were similar to those of the strain CEN.PK113-7D (ScURA1). An S. cerevisiae strain in which SjURA9 replaced ScURA1 also grew aerobically on SMUD and SMUD + ura but on both media showed an almost two-fold lower specific growth rate (0.19 h^− 1) than the other strains (Additional file 5; Table S1).

These results show that ArUra9, SjUra9 and DbUra9 are functional DHODs that, under aerobic conditions, complement a ura1Δ mutation in S. cerevisiae. Complementation of S. cerevisiae ura1 null mutants was previously demonstrated for Class-II DHODs of the oxygen-requiring yeasts Lachancea kluyveri (LkUra9 [19]) and Sch. pombe (SpUra3 [8]).

When expression cassettes for the heterologous URA9 genes were introduced in an S. cerevisiae ura1Δ strain on a multi-copy vector, specific growth rates of the resulting strains on SMUD as well as on SMUD + ura were lower (11–44%) than those of strains carrying a single integrated copy of the expression cassette (Additional file 5; Table S1).

Class II-DHODs of A. robustus, D. bruxellensis and Sch. japonicus support anaerobic growth of an S. cerevisiae ura1Δ strain. A. robustus, D. bruxellensis and Sch. japonicus have been reported to grow anaerobically in synthetic media without pyrimidine supplementation [26, 28, 41]. To test whether expression of ArUra9, DbUra9 and SjUra9 supports anaerobic, pyrimidine-prototrophic growth of S. cerevisiae, strains in which their structural genes replaced ScURA1 were grown anaerobically on SMUD and SMUD + ura.

The reference strain S. cerevisiae CEN.PK113-7D, which expresses the native fumarate-dependent Class I-A DHOD ScUra1, grew anaerobically at similar growth rates on SMUD and SMUD + ura (0.24–0.25 h^− 1, Additional file 5; Table S1), while strain IMK824 (ura1Δ) only grew anaerobically (0.26 h^− 1) on SMUD + ura (Additional file 5; Table S1). Also strains IMI446 and IMI447, in which ScURA1 was replaced by URA9 genes of the aerobic yeasts K. marxianus and O. parapolymorpha, did not show anaerobic growth unless media were supplemented with uracil (Additional file 5; Table S1). These results are in line with the coupling of canonical eukaryotic Class-II DHODs to the quinone pool of the mitochondrial respiratory chain [5, 8, 19, 42, 43], and with previous results of Gojković et al. [19] and Nagy et al. [8].

In contrast to expression of URA9 orthologs from aerobic yeasts, expression of Arura9 in S. cerevisiae supported fast anaerobic growth (0.25 h^− 1) without uracil supplementation (Additional file 5; Table S1). An S. cerevisiae strain in which ScURA1 was replaced by an SjURA9 expression cassette, also showed anaerobic growth on both SMUD and SMUD + ura, but at approximately two-fold lower specific growth rates (Additional file 5; Table S1). These results demonstrate that ArUra9 and SjUra9 function in S. cerevisiae under anaerobic conditions.

Strain IMI439, in which DbURA9 replaced ScURA1, did not show anaerobic growth on SMUD during the first 30 h of incubation (Additional file 5; Figure S1). After 68 h, when OD₆₀₀ had increased to 2.9, the strain was transferred to fresh SMUD, which resulted in immediate anaerobic growth. In a parallel experiment with strain IMI447, which expressed KmURA9, no growth was observed upon transfer to fresh SMUD.

These results indicated that not only the Class-II DHOD from D. bruxellensis [17, 27], but also those from the Neocallimastigomycete A. robustus and from the facultatively anaerobic yeast Sch. japonicus support pyrimidine synthesis under anaerobic conditions. The delayed anaerobic growth of an S. cerevisiae strain in which DbUra9 replaced ScUra1 suggested that anaerobic functionality of DbURA9 in S. cerevisiae may require physiological or genetic adaptations.

A cysteine residue in the active site of SjUra9 is required for activity under anaerobic conditions. In an attempt to identify potential biologically relevant differences in the amino acid sequences of Ura9 orthologs from oxygen-requiring yeast strains and the anaerobically functioning Ura9 enzymes, sequences of ArUra9, SjUra9, DbUra9, KmUra9 and OpUra9 were subjected to a multiple sequence alignment, along with those of the characterized Class II DHODs of L. kluyveri (LkUra9l), Sch. pombe (SpUra3), and DHOD sequences of the Neocallimastigomycetes N. californiae (NcUra9) and P. finnis (PfUra9) (Additional file 5; Figure S2). In comparison with the yeast Ura9 sequences, those of the three Neocallimastigomycetes showed a 76–81 amino-acid truncation at their N-termini. In canonical fungal Ura9 enzymes, the N-terminus contains a mitochondrial targeting sequence [5, 43] and is proposed to be involved in quinone binding [44, 45].

The Neocallimastigomycete Ura9 sequences as well as those of the two facultatively anaerobic yeasts (DbUra9 and SjUra9) contained a cysteine residue instead of the conserved serine that acts as catalytic base in canonical Class-II DHODs [11, 12, 46]. Of 331 fungal Ura9 orthologs (Additional file 1) only three additional proteins (from Coemansia reversa, Smittium culisis and Gonapodya prolifera) harbored a cysteine at this position, but did not show an N-terminal truncation (Additional file 1 and Additional file 5; Figure S2).

Since soluble fumarate- and NAD⁺-dependent Class-I DHODs also use a cysteine as catalytic base [12, 13], we investigated the relevance this residue for in vivo activity of ArUra9 and SjUra9 in S. cerevisiae. To this end, we introduced point mutations in Arura9 (C168S) and SjURA9 (C265S) to change the cysteine codon for a serine, yielding strains IMG007 (ura1Δ::Arura9^C168S) and IMG008 (ura1Δ::SjURA9^C265S). In addition, a point mutation in the corresponding serine codon of KmURA9 was introduced to change it to a cysteine, yielding strain IMG005 (KmURA9^S263C).

Changing the active-site serine residue (S263) of KmUra9 to a cysteine did not affect aerobic growth of S. cerevisiae IMG005 (ura1Δ::KmURA9^C263S) relative to that of its parental strain IMI446 (ura1Δ::KmURA9) (Fig. 3, Additional file 5; Table S1). This result indicated that, at least in KmUra9, the serine catalytic base that is strongly conserved in canonical Class-II DHODs, is not essential for activity under aerobic conditions. However, strain IMG005 did not grow anaerobically without uracil supplementation (Fig. 3, Additional file 5; Table S1), indicating that replacement of the catalytic-base serine residue by a cysteine is not sufficient to enable anaerobic functionality of KmUra9.

Replacing the active-site cysteine residue in ArUra9 by a serine did not affect aerobic or anaerobic growth on SMUD of strain IMG007 (ura1Δ::Arura9^C168S) relative to its parental strain, indicating that both variants were active under aerobic as well as anaerobic conditions (Fig. 3, Additional file 5; Table S1). This result demonstrated that the active-site cysteine residue in ArUra9 is not required for its functionality in anaerobic S. cerevisiae culture. A strikingly different result was obtained upon changing the cysteine residue in the active site of SjUra9 to a serine. Under aerobic conditions, exponential growth of strain IMG008 (ura1Δ::SjURA9^C265S) on SMUD was nearly two-fold slower than that of its parental strain IMI452 (ura1Δ::SjURA9). In contrast, strain IMG008 (ura1Δ::SjURA9^C265S) failed to grow on SMUD under anaerobic conditions. These results indicated that the active-site cysteine residue in SjUra9, but not in ArUra9, is required for DHOD activity under anaerobic conditions.

Subcellular localization of heterologous Ura9 orthologs expressed in S. cerevisiae. To investigate subcellular localization of Ura9 orthologs, eGFP fusions of anaerobically active ArUra9, DbUra9 and SjUra9, as well as OpUra9 were expressed from multicopy (mc) plasmids in an

S. cerevisiae ura1Δ strain, followed by fluorescence-microscopy analysis of the resulting strains (Fig. 4). A co-expressed mRuby2 fluorescent protein fused to the preCOX4 mitochondrial targeting sequence [47] was used as marker for mitochondrial localization.

In S. cerevisiae strains IME600 (ura1Δ mcArura9-eGFP), IME601 (ura1Δ mcDbURA9-eGFP) and IME604 (ura1Δ mcOpURA9-eGFP), mRuby2 fluorescence showed multiple small mitochondria, a pattern that is representative for respiring cells [48]. Consistent with the localization of canonical eukaryotic Class-II DHODs [5], OpUra9-eGFP fluorescence overlapped with that of preCOX4-MTS-mRuby2 (Fig. 4A). A similar co-localization of DbUra9-eGFP and mRuby in strain IME601 indicated that, despite its activity under anaerobic conditions, DbUra9 was targeted to mitochondria (Fig. 4B). In contrast, ArUra9-eGFP revealed a clear cytosolic localization (Fig. 4C), consistent with its N-terminal truncation, but representing a striking difference with canonical eukaryotic Class-II DHODs.

In strain IME602 (ura1Δ mcSjURA9-eGFP), mRuby2 fluorescence did not reveal the punctuate mitochondrial structures seen in the other strains. Instead, eGFP fluorescence was associated with tubular structures, that partially overlapped with a less defined mRuby2 fluorescence (Fig. 4D). Although elongated mitochondrial morphologies occur in fermenting S. cerevisiae cells [48], the diffuse mRuby2 fluorescence in strain IME602 did not allow for clear localization of SjUra9-eGFP. This strain was therefore also stained with the mitochondrial-membrane-potential dependent dye MitoTracker Deep Red. This approach only yielded vague tube-like structures or no fluorescence at all (Fig. 4E). Since the mRuby2-fused preCOX4-mitochondrial targeting sequence is also dependent on the mitochondrial membrane potential [47], we hypothesized that expression of SjURA9 reduces or abolishes mitochondrial membrane potential, possibly as a consequence of a loss of respiratory capacity [49].

Expression of SjURA9 in S. cerevisiae causes loss of respiratory capacity and mitochondrial DNA. To investigate whether SjURA9 causes loss of respiratory capacity, SjURA9-expressing

S. cerevisiae strains were tested for their ability to grow on non-fermentable carbon sources. In addition, presence of mitochondrial DNA was assessed by staining with the DNA-specific dye 4′,6-diamidino-2-phenylindole (DAPI, [50]) and by whole-genome sequencing.

In contrast to the reference strain S. cerevisiae CEN.PK113-7D (Fig. 5B), three independently constructed strains expressing SjURA9 (IMI452, IMI462 and IME571; Additional file 5; Table S4) failed to grow on synthetic medium supplemented with ethanol and glycerol (SMEG, Fig. 5E, F, G, H, respectively). Removal of the SjURA9 expression plasmid from strain IME571, yielding strain IMS1206, did not recover growth on these non-fermentable carbon sources (Fig. 5I). The inability of SjURA9-expressing strains to grow on SMEG resembled that of the respiratory-deficient strain IMK242 [51]. In contrast, strain IME603, which expressed ScURA1 from a multicopy plasmid, as well as all other URA9-expressing ura1Δ strains did grow on SMEG, (Additional file 5; Figure S2).

Sch. japonicus strains are naturally respiratory deficient but do have a mitochondrial genome [52]. Extranuclear DAPI staining of Sch. japonicus cultures revealed tubular structures which probably represent mitochondria (Fig. 5A). In contrast, reference S. cerevisiae strain CEN.PK113-7D (Fig. 5B) and an S. cerevisiae strain expressing ScURA1 from a multicopy plasmid (IME603; Fig. 5C), showed punctuated extranuclear DAPI staining. Three of the four SjURA9-expressing S. cerevisiae strains only clearly showed fluorescent nuclei, while strain IMI452 (ura1Δ::SjURA9) showing vague DAPI-stained tubular structures. Whole-genome sequencing of the SjURA9-expressing S. cerevisiae strains IMI452, IMI462, IMG008 and IME571 indicated absence of mitochondrial DNA in all four strains (Fig. 5). Consistent with this observation, respiratory capacity was not restored when the SjURA9-expressing plasmid was removed from strain IME571 (Fig. 5).

ArUra9 can use free FAD and FMN as electron acceptors. The cytosolic localization of ArUra9 and the absence of mitochondria in Neocallimastigomycetes implied that its activity in S. cerevisiae was unlikely to involve components of the mitochondrial respiratory chain. To identify possible natural electron acceptors of ArUra9, enzyme-activity assays were performed in cell extracts of strain IME569 (ura1Δ mcArura9). Cell extracts of strains IME603 (mcScURA1) and IMK824 (ura1Δ) were included as references.

Consistent with literature reports [53], cell extracts that only contained the Class-I DHOD ScUra1 (strain IME603) showed dihydroorotate oxidation with fumarate as electron acceptor (0.11 µmol∙mg protein^− 1.min^− 1), while a similar activity was observed with the artificial electron acceptor phenazine methosulfate (PMS, Table 2). ScUra1-containing cell extracts also showed dihydroorotate oxidation without addition of electron acceptor (Table 2), which was attributed to a previously reported ability of DHODs to use molecular oxygen as electron acceptor [54–57].

Table 2

**Dihydroorotate-dehydrogenase activities in cell extracts of** S. cerevisiae **strains expressing different DHODs, measured with different electron acceptors.** Activities were measured in *S. cerevisiae* strains IMK824 (*ura1Δ*), IME603 (expressing *ScURA1* from a multicopy plasmid) and in the strain IME569 (expressing *Arura9* from a multicopy plasmid). Activities were measured without addition of electron acceptor (none), 0.1 mM phenazine methosulfate (PMS), 1 mM fumarate, 1 mM nicotinamine adenine dinucleotide (NAD⁺), 1 mM nicotinamide adenine dinucleotide phosphate (NADP⁺), 0.1 mM decylubiquinone (Q_D), 20 µM flavin mononucleotide (FMN) or 20 µM flavin adenine dinucleotide (FAD). Activities are represented as the average ± mean deviation of activities measured with two independently prepared cell extracts.
Electron acceptor
	S. cerevisiae IMK824 ura1Δ	S. cerevisiae IME603 mcURA1	S. cerevisiae IME569 ura1Δ mcArura9
-	< 0.005	0.010 ± 0.001	0.008 ± 0.001
PMS	< 0.005	0.111 ± 0.007	0.132 ± 0.029
Fumarate	< 0.005	0.110 ± 0.020	0.006 ± 0.000
NAD⁺	< 0.005	< 0.005	< 0.005
NADP⁺	< 0.005	< 0.005	< 0.005
Q_D	< 0.005	0.033 ± 0.022	0.009 ± 0.003
FMN	< 0.005	0.037 ± 0.002	0.126 ± 0.012
FAD	< 0.005	0.011 ± 0.002	0.148 ± 0.032

A high rate of PMS-dependent dihydroorotate oxidation (0.13 µmol∙mg protein^− 1.min^− 1) confirmed DHODase activity in cell extracts of the ArUra9-expressing strain IME569. A low activity in the absence of an added electron acceptor (Table 2) suggested that, like other DHODs, ArUra9 can use molecular oxygen as electron acceptor. Assays in which fumarate, NAD⁺, NADP⁺ or decylubiquinone were added to reaction mixtures did not increase activities beyond this basal level. Other compounds with a standard redox potential above or close to that of DHOD-bound FMN cofactors (-242 to -310 mV; [57–59]) were therefore tested. Addition of flavin adenine mononucleotide (FAD; E’⁰ = -219 mV [60]), flavin mononucleotide (FMN; E’⁰ = -219 mV [60]) strongly promoted dihydroorotate oxidation by cell extracts of the ArUra9-expressing strain IME569, with DHOD activities of 0.15 µmol∙mg protein^− 1·min^− 1 and 0.13 µmol∙mg protein^− 1·min^− 1, respectively. Supplementation of dihydroxyacetone phosphate (DHAP), acetaldehyde, pyruvate or oxaloacetate as electron acceptor did not increase enzyme activity.

Fast anaerobic growth of DbUra9-dependent S. cerevisiae strains correlates with mutations in FUM1. Attempts to identify electron acceptors of the mitochondrial DbUra9 and SjUra9 enzymes by experiments with cell extracts and isolated mitochondria were unsuccessful. We therefore investigated whether the adaptation of S. cerevisiae IMI439 (ura1Δ::DbURA9) to fast anaerobic, pyrimidine-prototrophic growth (Additional file 5; Figure S1) could provide insight into its in vivo cofactor use. Two independent cultures of this strain on SMUD were incubated anaerobically until growth occurred and then transferred to fresh medium, in which they instantaneously grew (Additional file 5; Figure S1). Upon reaching stationary phase, two single colonies were isolated from each culture, yielding isolates IMS1167, IMS1168, IMS1169 and IMS1170. To check whether these isolates had acquired stable mutations that enabled anaerobic growth, they were first grown aerobically on SMUD + ura and then transferred to anaerobic medium with a reduced uracil content (SMUD + ura0.1). Upon reaching late-exponential phase, the resulting anaerobic, pyrimidine-limited precultures were used to inoculate anaerobic cultures on SMUD and SMUD + ura, in which all four isolates showed instantaneous anaerobic growth (Additional file 5; Table S1). To identify possible causal mutation(s) involved in this acquired phenotype, the genomes of the four strains were sequenced and compared with that of their parental strain IMI439 (ura1Δ::DbURA9). Strains IMS1167 and IMS1168, which originated from the same anaerobically adapted culture, both harbored the same two mutations (resulting in the amino-acid changes Vps1^I410L and Fum1^M432I; Table 3). Strains IMS1169 and IMS1170, which were isolated from the second anaerobically adapted cultures, each harbored a different mutation in FUM1 (resulting in the amino-acid changes Fum1^A294V and Fum1^T218M, respectively; Table 3). The occurrence of three different mutations in FUM1, which encodes fumarase, suggests that the intracellular fumarate concentration may be important for the activity of DbUra9 in anaerobic

Table 3

**Mutations in** DbURA9 **expressing strains, evolved for anaerobic pyrimidine prototrophy.** Two cultures of IMI439 (*ura1Δ::DbURA9*) were independently evolved under anaerobic conditions on SMUD (Figure S1). Two single colony isolates from each flask were subjected to whole-genome resequencing and predicted amino-acid substitutions were only found in Vps1 and Fum1.
Evolution line	Strain	Mutations
Evolution line	Strain	Vps1	Fum1
Flask 1	IMS1167	I410L	M432I
Flask 1	IMS1168	I410L	M432I
Flask 2	IMS1169	-	A294V
Flask 2	IMS1170	-	I218M

S. cerevisiae cultures.

Fumarate-dependent Class-IA DHODs in S. cerevisiae (ScUra1) and closely related yeasts circumvent the oxygen requirement of respiration-dependent pyrimidine synthesis via the mitochondrial Class-II DHODs that occur in most other fungi [19, 20, 22]. The assumption that presence of a Class-IA DHOD is required for anaerobic pyrimidine prototrophy in fungi was first called into question when analysis of the genome of Dekkera bruxellensis showed absence of a Class-IA DHOD gene and, instead, revealed a sequence (DbURA9) with similarity to Class-II DHOD genes [17, 23, 27]. We identified similar situations in obligately anaerobic Neocallimastigomycetes and in the facultatively anaerobic fission yeast Sch. japonicus (Table 1, Fig. 1). Heterologous expression studies in ura1Δ S. cerevisiae showed that DbUra9, as well as orthologs from the Neocallimastigomycete Anaeromyces robustus (ArUra9) and Sch. japonicus (SjUra9), supported anaerobic pyrimidine prototrophy. This phenotype was not observed for ura1Δ strains expressing URA9 genes from the aerobic yeasts Kluyveromyces marxianus and Ogataea parapolymorpha (Fig. 2) nor in similar experiments involving URA9 genes from other aerobic yeasts [8, 19, 25].

A cysteine as catalytic base is considered a hallmark of Class-I DHODs [56] while, with few reported exceptions, Class-II enzymes have a serine in this position [16]. Instead, the three anaerobically active Ura9 orthologs investigated in this study had a Cys in the catalytic-base position and shared this feature with only 3 of 331 other predicted fungal Ura9 orthologs. The latter three sequences originated from Coemansia reversa, Smittium culicis and Gonapodya prolifera (Fig. 1), which were all originally isolated from microaerobic or anoxic environments (dung [61], insect guts [62] and submerged fruits [63], respectively). A Cys-to-Ser change in the active site of Sch. japonicus SjUra9 specifically abolished its ability to support anaerobic pyrimidine prototrophy of S. cerevisiae (Fig. 3). While this result suggested that a Cys as catalytic base can be relevant for anaerobic functionality, changes at the same position of

K. marxianus KmUra9 (Ser to Cys) and A. robustus ArUra9 (Cys to Ser) showed it is neither sufficient nor absolutely required for anaerobic activity of Class-II DHODs (Fig. 3).

Consistent with the N-terminal truncation of Ura9 orthologs from Neocallimastigomycetes (Additional file 5; Figure S2), an ArUra9-eGFP fusion expressed in S. cerevisiae was localized to the cytosol (Fig. 4). Enzyme assays in cell extracts showed that ArAro9 can use free FAD and FMN as electron acceptors (Table 2), which was not previously observed for DHODs. Biochemical standard redox potentials of the non-enzyme-bound FADH₂/FAD and FMNH₂/FMN redox couples (E⁰’ = -219 mV; [64]) and those of DHOD-bound FMNH₂/FMN (-242 to -330 mV; [57–59]) are compatible with either of these flavin cofactors acting as physiological electron acceptor of ArAro9. In anaerobic chemostat cultures of S. cerevisiae, intracellular FAD and FMN contents of 0.17 and 0.09 µmol (g biomass)⁻¹ were reported [65]. While use of free flavins in cellular redox reactions is relatively rare, the S. cerevisiae fumarate-reductase Osm1 can re-oxidize free FADH₂ [66]. Since combined deletion of OSM1 and its paralog FRD1 abolishes anaerobic growth of S. cerevisiae [67], we could not experimentally verify their involvement in the in vivo anaerobic activity of ArUra9. Sequences with strong homology to S. cerevisiae Frd1 and/or Osm1 were found in Neocallimastigomycetes, Sch japonicus and D. bruxellensis (Additional file 5; Table S3). In silico prediction of subcellular localization indicated that these putative fumarate reductases were mitochondrial in Sch. japonicus and D. bruxellensis. Consistent with the inferred cytosolic localization of ArUra9, they were predicted to occur in the cytosol of Neocallimastigomycetes (Additional file 5; Table S3).

Previous studies implicated the N-terminal domains of Class II DHODs in quinone binding [44, 68]. Based on analysis of 1500 Class II DHOD sequences and structural alignments, Sousa et al. [16] proposed conserved residues involved in quinone binding, stabilizing and pocket entry (Additional file 5; Figure S2). Ura9 sequences from Neocallimastigomycetes lacked several residues proposed to be involved in quinone binding due to their N-terminal truncation, while for another, a positively-charged residue was replaced by a hydrophobic one (Additional file 5; Figure S2). Although quinones have been found in Neocallimastigomycetes [69], these observations and the absence of quinone-dependent DHOD activity in cell extracts containing ArUra9 (Table 2) indicate that they are unlikely to be involved in pyrimidine synthesis.

Sch. japonicus and D. bruxellensis Ura9 orthologs retained an N-terminal mitochondrial targeting sequence, as well as conserved residues proposed to be involved quinone binding (Additional file 5; Figure S2). The only difference in quinone-associated residues was a Tyr-to-Phe change in SjUra9 (Y137), which may not have affected functionality (Additional file 5; Figure S2). We were unable to measure activities of DbUra9 and ScUra9 in cell extracts with the artificial electron acceptor PMS or other potential electron acceptors.

DbUra9-expressing ura1Δ isolates of S. cerevisiae evolved for fast anaerobic pyrimidine-prototrophy revealed three different mutations in the FUM1 fumarase gene. Mutations in the human fumarase gene (FH) have been implicated with different types of cancer due to increased fumarate concentrations [70]. Thr218 and Ala294 of DbUra9 correspond to Val197 and Ala274, respectively, in FH and are both located in highly conserved regions [71, 72], while Ala274 resides in the active site [73]. Mutation of Ala274 to a valine in human fumarate hydratase (FH) was implicated in ovarian mucinous cystadenoma [74], and resulted in a 50% decreased activity of the enzyme [72]. By analogy, it seems probably that the Fum1^A294V change in strain IMS1169 also led to a reduced fumarase activity. Corresponding amino acids in human FH of Thr218 and Met432 are located in the core helix, and accumulated fumarate resulting from mutations in this helix have been associated with different types of cancer [73]. Higher intracellular fumarate concentrations due to a reduced activity or affinity of Fum1 variants might favor in vivo use of fumarate as electron acceptor, as in Class I-A enzymes [56]. Alternatively, they could stimulate reoxidation of FADH₂ via fumarate reductases.

Anaerobic pyrimidine synthesis, combined with acquisition by horizontal gene transfer of genes enabling sterol-independent anaerobic growth (squalene hopene cyclase; [29]) and anaerobic deoxynucleotide synthesis (Class-I ribonucleotide reductase; [37]), indicates that Sch. japonicus is remarkably well adapted to anaerobic growth. The observation that independently constructed SjUra9-expressing S. cerevisiae strains all showing loss of respiratory capacity and loss of mitochondrial DNA (Fig. 5) revealed a trade-off between anaerobic pyrimidine synthesis and respiratory competence. Quinone-dependent DHODs are known to react with oxygen, leading to formation of hydrogen peroxide and superoxide [54]. In S. cerevisiae, these reactive oxygen species have been implicated in loss of respiratory capacity [75]. Despite presence of respiratory proteins, including low levels of all cytochromes [76], Sch. japonicus strains are naturally respiratory deficient [77–79]. Based on our results, this phenotype may either have provided a driving force for evolution of a respiration-independent DHOD or, alternatively, be a consequence of the adaptation of SjUra9 for anaerobic functionality.

Our results show that, in addition to the well-established acquisition of a Class-I DHOD by S. cerevisiae and closely related yeasts, at least three separate events in fungal evolution enabled anaerobic pyrimidine synthesis by variants of Class-II DHODs that do not depend on aerobic respiration. These anaerobically active variants were shown to have a cysteine instead of a conserved serine residue in their catalytic sites. Their in vivo activities were not dependent on aerobic respiration, and, in Neocallimastigomycetes, they were not membrane associated and could use free FAD or FMN as electron acceptor. These remarkable differences with canonical Class-II DHODs underline the plasticity of fungal genomes and genes under selective pressure and extend our knowledge on eukaryotic adaptation to anoxic environments. We hope this study will stimulate further research to elucidate the mechanism(s) by which the reduced flavin cofactor of the membrane-associated Class-II DHODs from D. bruxellensis and Sch. japonicus is re-oxidized under anaerobic conditions and the trade-off between anaerobic functionality and loss of respiratory capacity in the case of SjUra9.

Yeast strains, media and strain maintenance. Saccharomyces cerevisiae strains were derived from the CEN.PK lineage (Additional file 5; Table S4; [80,81]). Ogataea parapolymorpha CBS11895, Kluyveromyces marxianus CBS6556 and Schizosaccharomyces japonicus CBS5679 were obtained from the Westerdijk Institute (Utrecht, The Netherlands). Synthetic media with ammonium as nitrogen source (SM) and with urea as nitrogen source (SMU), containing vitamins and trace elements, were prepared and sterilized as described previously [82,83]. A separately autoclaved (30 min, 110 °C) d-glucose solution (50% w/v) was added to sterile SMU or SM at a concentration of 20 g L^‑1, yielding SMUD and SMD, respectively. SM with ethanol and glycerol (SMEG) and complex yeast extract-peptone-glucose medium (YPD) were prepared as described previously [84]. Where indicated, YPD was supplemented with 200 mg L^-1filter-sterilized geneticin (G418; Thermo Fischer Scientific, Waltham, MA) or hygromycin B (HygB; Thermo Fischer Scientific). Synthetic media for anaerobic growth experiments were supplemented with Tween 80 and ergosterol [82]. Uracil-auxotrophic strains were routinely grown on SMUD supplemented with 150 mg L^-1 uracil (SMD+ura) or, to obtain uracil-limited pre-cultures, with 15 mg L^-1 uracil (SMUD+ura0.1). Solid synthetic and complex media were prepared by adding 20 g L^-1 Bacto agar (Difco laboratories Inc, Detroit, MI) prior to autoclaving. Escherichia coli strains were grown on Lysogeny Broth (LB; [85]), supplemented with 100 mg L^-1 filter-sterilized ampicillin (Sigma Aldrich, St. Louis, MO) or chloramphenicol (Sigma Aldrich) as indicated (LB-amp and LB-cam, respectively). Frozen stock cultures of yeast strains were prepared as described previously [86] after growth to mid-exponential phase at 30 °C on YPD (strains CENPK.113-5D, CEN.PK113-7D, IMX585, IMX2600, CBS6556 and CBS11895), on SMUD+ura (strain IMK824) or on SMUD (other strains). E. coli strains were grown at 37 °C on LB-amp or LB-cam and frozen stock cultures were prepared as described by Mans et al. [86].

Molecular biology techniques. Phusion High Fidelity DNA Polymerase (Thermo Fischer Scientific) and PAGE-purified oligonucleotide primers (Additional file 5; Table S5, Sigma Aldrich) were used in polymerase chain reactions (PCRs) for cloning and sequencing. Diagnostic PCRs were performed with desalted oligonucleotides (Additional file 5; Table S5, Sigma Aldrich) and DreamTag Mastermix 2X (Thermo Fisher Scientific). Genomic DNA as template for PCRs was isolated using a YeaStar Genomic DNA kit (Zymo Research, Irvine, CA). PCR products were purified with a GeneElute PCR Clean-Up kit (Sigma Aldrich) or from 1% agarose gels using a Zymoclean Gel DNA Recovery Kit (Zymo Research). Gibson Assembly of purified DNA fragments with 20 bp sequence overlaps was performed with the NEBuilder HiFi DNA Assembly Mastermix (New England Biolabs, Ipswich, MA). Golden-Gate assembly was performed according to Lee et al. [87]. E. coli XL1-Blue (Agilent Technologies, Santa Clara, CA) was chemically transformed following manufacturer’s instructions and plated on selective media. Correct plasmid assembly was verified by diagnostic PCRs on E. coli transformants [86]. Cas9-mediated genome editing in S. cerevisiae was performed according to Mans et al. [86]. The LiAc/SS-DNA/PEG method [88] was used for yeast transformation with plasmids (at least 1 μg DNA per transformation) or linear DNA fragments (0.5-1 μg per transformation). Single-colony isolates of randomly picked transformants were obtained by three re-streaks on selective media. Integrations and deletions were verified by diagnostic PCRs with genomic DNA as a template. Construction of plasmids and yeast strains is described in detail in Additional file 6.

Whole-genome DNA sequencing. Genomic DNA of yeast strains was isolated from overnight cultures on YPD, except for strain IME571 that was grown on SMUD, using the Qiagen Genomic DNA 100/G kit (Qiagen, Hilden, Germany) with the Proteinase-K step extended to 3 h. DNA concentrations were measured on a Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA) with the QuBit BR dsDNA Assay kit (Invitrogen). Whole-genome sequencing on an Illumina MiSeq platform (Illumina Novoseq 6000, Illumina Inc., San Diego, CA, USA) was performed by Novogene Europe (Cambridge, UK; strains IMI439, IMS1167, IMS1168, IMS1169 and IMS1170), Macrogen Europe BV (Amsterdam, The Netherlands; strains IMG008, IME571 and IMS1206) or in-house (strains IMI452 and IMI462). For in-house sequencing, the Nextera DNA Flex 509 Library Prep kit (Illumina) was used for paired-end library preparation. Genome sequences were deposited at GenBank (BioProject accession number PRJNA745202).

Whole-genome sequence analysis. Sequencing data were processed as described previously [89]. For sequence analysis of the evolved strains IMS1167-IMS1170, their parental strain IMI439 was used as reference to obtain sequence differences. For IMI452/IMI462/IMG008 or IME571/IMS1206 IMI452 and IME571 were used as reference, respectively. Identified SNP’s were individually checked with the Integrated Genomics Viewer (IGV; [90]). Mitochondrial DNA coverage plots were generated by calculating the average of non-overlapping 500 bp sliding windows (R version 3.6.0).

Protein sequence homology search and phylogenetic analysis. Proteomes of Anaeromyces robustus (NCBI taxid 1754192), Piromyces finnis (taxid 1754191), Neocallimastix californiae (taxid 1754190), Schizosaccharomyces japonicus (taxid 402676), D. bruxellensis (5007; [91]), K. marxianus (1003335) and O. parapolymorpha (871575) were subjected to protein blast search in NCBI [92], using LkUra1 (UniProt KB accession number Q7Z892) and LkUra9 (Q6V3W9) as queries and applying default settings. Similarly, the proteomes of A. robustus, P. finnis, N. californiae, Sch. japonicus and that of Dekkera bruxellensis were subjected to a protein blast using ScFrd1 (GenBank accession number EIW10990.1) or Osm1 (EIW09573.1) as query, default settings were applied.

To predict the localization of putative fumarate reductases in A. robustus, N. californiae, P. finnis, D. bruxellensis and Sch. japonicus, protein sequences were compared to known fungal sorting signals and motifs using the online computational tool WoLF PSORT [93]. The highest scoring cellular component for fungal settings was retrieved.

Bacterial and fungal amino acid sequences available from UniProt reference proteomes release 2019_02 [94] were systematically searched for Class-II dihydroorotate dehydrogenase orthologs. The database of fungal reference proteomes (taxid 4751) was supplemented with sequences available from the UniProt trembl division for the following organisms: Dekkera bruxellensis (taxid 5007), Komagataella phaffii (taxid 981350), Komagataella pseudopastoris (taxid 169507), Komagataella pastoris (taxid 4922), Ogataea polymorpha (taxid 460523), Pichia membranifaciens (taxid 763406), Pichia kudriavzevii (taxid 4909), Neocallimastix californiae (taxid 1754190), Piromyces finnis (taxid 1754191), Anaeromyces_robustus (taxid 1754192) and Piromyces sp. E2 (taxid 73868). Then, a Class-II DHOD of Lachancea kluyveri CBS3082 (Q6V3W9; LkUra9)[19] was used as query for a HMMER search [35], using cutoff values of 1e-5 and requiring hits to correspond to at least 75% of the query sequence length resulting in 724 fungal and 1595 bacterial Ura9 homologs. The sets of Ura9 homologs were further used to select a set of orthologs. For this purpose, the database of fungal proteomes was used to calculate all possible co-ortholog sets with proteinortho v6.0.25 [95] running diamond v2.0.8 [96], obtaining 331 Ura9 orthologs (Additional file 1). Similarly, the search for bacterial Ura9 orthologs resulted in 73 sequences (Additional file 1). Ura9-orthologous amino-acid sequences were then subjected to multiple sequence alignment using MAFFT v7.40286 [97] in “einsi” mode. Alignments were trimmed using trimAl v1.287 [98] in “gappyout” mode, and used to build a phylogenetic tree with RAxML-NG v0.8.188 [99] using 10 random and 10 parsimony starting trees, 100 Felsestein Bootstrap replicates, and LG model. The resulting phylogenetic tree was visualized using iTOL (Interactive Tree Of Life) tool v6 [100].

Multiple sequence alignment of selected Ura9-orthologs was performed in Clustal Omega [101] with default settings. Protein sequences of Ura9 orthologs were retrieved from the Uniprot database for Sch. pombe (SpUra3; Uniprot KB accession number P32747); L. kluyveri (LkUra9; Q6V3W9), O. parapolymorpha (OpUra9; W1QJ07), K. marxianus (KmUra9; Q6SZS6), E. coli (EcUra9; P0A7E1), D. bruxellensis (DbUra9; I2JUI3), Sch. japonicus (SjUra9; B6JXQ5), A. robustus (ArUra9; A0A1Y1XN91), N. californiae (NcUra9; A0A1Y2ELQ6) and P. finnis (PfUra9; A0A1Y1VDI5) and Coemansia reversa (CmUra9; A0A2G5BHD4), Smittium culicis (ScuUra9; A0A1R1YI62) and Gonopodya prolifera (GpUra9; A0A139AY32).

Cultivation of yeast strains. Aerobic shake-flask cultures were grown in an Innova Incubator (New Brunswick Scientific, Edison, NJ) at 30 °C and 200 rpm. Pre-cultures in 100-mL shake flasks with a working volume of 40 mL were inoculated with frozen stock cultures. Primary pre-cultures of yeast strains expressing plasmid-borne DHOD genes were grown on SMUD and those of other yeast strains on SMUD+ura, and were used to inoculate a secondary pre-culture on SMUD (for plasmid expressing strains) or SMUD+ura0.1. Upon reaching late exponential phase, cultures were centrifuged (5 min at 3000 x g) and washed twice with demineralized water. Washed cell suspensions were used to inoculate 500-mL shake flasks containing 100 mL of SMUD+ura or SMUD, at an initial optical density at 660 nm (OD₆₆₀) of 0.2.

Anaerobic cultures were grown in 100-mL shake flasks with a working volume of 80 mL. Precultures were grown aerobically on SMUD+ura as described above, until stationary phase, washed twice with sterile demineralized water and transferred to an anaerobic preculture on SMUD+ura0.1, supplemented with Tween80 and ergosterol. Flasks were incubated on an IKA KS 260 orbital shaker (240 rpm; Dijkstra Verenigde BV, Lelystad, The Netherlands) placed in a Shel Lab Bactron 300 anaerobic workstation (Sheldon Manufacturing Inc, Cornelius, OR) at 30 °C. The gas mixture supplied to the anaerobic workstation contained 10% CO₂, 5% H₂ and 85% N₂.Measures to minimize inadvertent oxygen entry were implemented as described by [102]. When anaerobic precultures reached stationary phase, they were used to inoculate cultures on SMUD and SMUD+ura supplemented with Tween 80 and ergosterol.

For spot-plate experiments, yeast strains were pre-grown on 20 mL SMUD in 100-mL shake flasks, centrifuged (5 min, 3000 g) and washed twice with demineralized water. Washed cultures were used for cell counts with a Z2 Coulter particle count and size analyzer (Beckman Coulter, Brea, CA) set at particle size 2.5-7.5 μm. Cells were diluted to a final concentration of 2.5∙10⁵ cells mL^-1, and subsequently diluted to 2.5∙10⁴ cells mL^-1, 2.5∙10³ cells/mL and 2.5∙10² cells mL^-1. From these four dilutions, 4 μL of each strain and dilution was transferred to SMD, SMD+ura, SMEG and SMEG+ura plates. All strains were pregrown and plated in duplicate.

Analytical methods. Extracellular metabolite concentrations were measured by high performance liquid chromatography as described by Verhoeven et al. [103] Optical density at 660 nm of aerobic cultures was measured using an Jenway 7200 spectrophotometer (Bibby Scientific, Staffordshire, UK) after accurate dilution to an OD₆₆₀ between 0.1 and 0.3. Anaerobic cultures were first diluted to an optical density at 600 nm (OD₆₀₀) between 0.15 and 0.35, followed by optical densities measurements at 600 nm on an Ultrospec 10 cell density meter (Biochrom, Harvard Biosience, Holliston, MA) that was placed in the anaerobic workstation [102].

Microscopy analysis and staining. MitoTracker Deep Red FM (Invitrogen) staining was performed on early exponential phase aerobic cultures by adding 250 nM MitoTracker Deep Red FM to a 1-mL culture sample and subsequent incubation in the dark at 37 °C for 15 min. DNA staining was performed on 1-mL samples of early exponential phase, aerobic cultures on 10 mL SMUD in 50-mL vented Greiner tubes (Greiner Bio-One, Kremsmünster, Austria). Cultures were supplemented with 300 nM 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride (Sigma Aldrich) and incubated in the dark for 10-15 min at 20 °C. Phase-contrast microscopy was performed using a Zeiss Axio Imager Z1 (Carl Zeiss AG, Oberkochen, Germany) that was equipped with a HAL 100 Halogen illuminator, HBO 100 illuminating system and AxioCam HRm Rev3 detector (60N-C 1’’ 1.0x) (Carl Zeiss AG). The lateral magnification objective 100x/1.3 oil was used with Immersol 518F type F immersion oil (Carls Zeiss AG). Fluoresence of eGFP was detected using filter set 10 (Carl Zeiss AG; excitation bandpass (BP) 470/20, emission 540/25). MitoTracker Deep Red and mRuby2 were imaged with filterset 14 (excitation BP 535/25, emission longpass (LP) 590) and 50 (excitation BP 640/30, emission BP 690/50) respectively (Carls Zeiss AG). For analysis of DAPI dihydrochloride fluorescence, filterset 49 (excitation Short Pass (SP) 380, emission BP 445/50) was used (Carl Zeiss AG). Results were analysed using the Fiji package of ImageJ [104].

Preparation of cell extracts. Strains carrying multi-copy plasmids expressing dihydroorotate dehydrogenases, were grown to mid-exponential phase in 100-mL shake flask cultures on SMUD. After centrifugation at 3000 x g and at 0 °C, biomass was resuspended in 4 mL ice-cold 10 mM potassium phosphate buffer (pH 7.5) with 2 mM EDTA and stored at -20 °C. Samples were thawed on ice, centrifuged at 3000 x g and at 4 °C, washed with 10 mL ice-cold sonication buffer (100 mM potassium phosphate buffer, pH 7.5 with 2 mM MgCl₂) and resuspended in 4 mL sonication buffer containing 1 tablet of cOmplete Mini protease inhibitor (Sigma Aldrich) per 10 mL buffer. Cell extracts were prepared by sonication and centrifugation as described previously [105]. Bovine serum albumin (Sigma Aldrich) was used as a reference for analyzes of protein concentrations in cell extracts [106].

Dihydroorotate dehydrogenase activity assays in cell extracts. Dihydroorotate dehydrogenase assays were performed at 30 °C in potassium phosphate buffer, (100 mM, pH 7.5) using a Hitachi U-3010 UV/Visible spectrophotometer (Chiyoda, Tokyo, Japan). Formation of orotate or reduction of NAD(P)⁺ was monitored by measuring absorbance at 300 nm (ε = 3.05 mM^-1 cm^-1; [14]) or 340 nm (ε = 6.22 mM^-1 cm^-1; [107]) respectively, upon addition of 1 mM dihydroorotate to a temperature-equilibrated reaction mixture containing buffer, cell extract and/or either of the electron acceptors fumarate (1 mM), decylubiquinone (Q_D, 0.1 mM, dissolved in dimethylsulfoxide), nicotinamide adenine dinucleotide (NAD⁺, 1 mM), nicotinamine adenine dinucleotide phosphate (NADP⁺, 1 mM), flavine adenine dinucleotide (FAD, 20 μM), flavin mononucleotide (FMN, 20 μM) or the artificial electron acceptor phenazine methosulfate (PMS, 0.1 mM). Enzyme assays were performed on two separately prepared cell extracts. Reduction potentials of tested electron acceptors mentioned in the text are relative to the standard hydrogen electrode.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

Figure 1 was made available online

(https://itol.embl.de/export/193190253145446711626368835)

Whole-genome sequencing data from strains IMS1167, IMS1168, IMS1169, IMS1170, IMI452, IMI462, IMG008, IME571 and IMS1206 was deposited at NCBI (BioProject accession number PRJNA745202)

Competing interests

We declare no conflicts of interest

Funding

This work was funded by an Advanced Grant of the European Research Council to JTP (grant 555 694633).

Authors' contributions

JB and JTP wrote the initial version of the manuscript. All authors critically read the manuscript, provided input and approved the final version. All authors contributed to the experimental design. JB, CCK and MAHL performed microscopy analysis, enzyme assays were performed by JB and MAHL Sequence analysis was performed by MB and JB and phylogenetic analysis by RAOM. Strain construction was performed by JB and MW and all growth experiments were performed by JB,

Acknowledgements

We gratefully thank Göktuğ Aba for construction of pUDC286 and Siem Eerden for construction of pUDE672 and pUDE677, Robert Stella for pUD538 and Petrik Buitenhuis for construction of pUDE815. We acknowledge Jisk van der Meer for constructing pUDR386, pUDE765, pUDE809, IMK839, IMI446, IMI447 and IMI452 and Magdalena Kaminska for construction of IMX2165, IMX2203, IMX2209, IMG005 and IMG007. We thank Marc Strampraad for the help with the enzyme assays and Sanne Wiersma and Nicole Bennis for their help with the aerobic growth study. We also thank Sanne Wiersma for critically reading the manuscript.

Ar Anaeromyces robustus

Db Dekkera bruxellensis

DHAP Dihydroxyacetone phosphate

DHOD Dihydroorotate dehydrogenase

Cr Coemansia reversa

Cys Cysteine

FAD Flavin adenine dinucleotide

FH Fumarate hydratase

FMN Flavin mononucleotide

GFP Green fluorescent protein

Gp Gonapodya prolifera

HGT Horizontal gene transfer

Km Kluyveromyces marxianus

Nc Neocallimastix californiae

Op Ogataea parapolymorpha

Pf Piromyces finnis

PMS phenazine methosulfate

Q_D decylubiquinone

Sc Saccharomyces cerevisiae

Scu Smittium culicus

Ser Serine

SMD synthetic dextrose medium

SMEG synthetic ethanol/glycerol medium

SMUD synthetic urea medium with dextrose

Sj Schizosaccharomyces japonicus

μ maximum specific growth rate

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Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis

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