Identification of oxygen-independent pathways for pyridine-nucleotide and Coenzyme-A synthesis in anaerobic fungi by expression of candidate genes in yeast

Perli, Thomas; Vos, Aurin M.; Bouwknegt, Jonna; Dekker, Wijb J C; Wiersma, Sanne J; Mooiman, Christiaanl; Ortiz‐Merino, Raúl A.; Daran, Jean‐Marc; Pronk, Jack T.

doi:10.1101/2020.07.06.189415

Cited by 3 publications

(3 citation statements)

References 76 publications

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“…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.…”

Section: Methodsmentioning

confidence: 99%

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

Bouwknegt

Koster

Vos

et al. 2021

Fungal Biol Biotechnol

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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. A strain expressing DbURA9 showed delayed anaerobic growth without pyrimidine supplementation. Adapted faster growing DbURA9-expressing strains showed mutations in FUM1, which encodes fumarase. 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. Expression of SjURA9 in S. cerevisiae reproducibly led to loss of respiratory competence and mitochondrial DNA. 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.

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Section: Methodsmentioning

confidence: 99%

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

Bouwknegt

Koster

Vos

et al. 2021

Fungal Biol Biotechnol

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“…Furthermore, genes related to fatty liver diseases, such as fabp1b . 1, adcal ( 57 ), fabp2 ( 56 ), apoa1a ( 58 ), cd36 ( 59 ) and acox1 ( 60 ), were highly expressed in cluster 5 (Figure 2D), suggesting that cluster 5 may also be involved in the formation of fatty liver.…”

Section: Resultsmentioning

confidence: 99%

Single-cell transcriptome landscape of zebrafish liver reveals hepatocytes and immune cell interactions in understanding nonalcoholic fatty liver disease

Huang

Liu

Wang

et al. 2022

Preprint

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Zebrafish have emerged as an attractive animal model for studying nonalcoholic fatty liver disease (NAFLD). However, little is known about the cell types and intercellular interactions in zebrafish liver. Here, we established a liver atlas that consists of 10 cell types using single-cell RNA sequencing. By examining the heterogeneity of hepatocytes and analyzing the expression of NAFLD-associated genes in the specific cluster, we provide a potential target cell model to study NAFLD. Additionally, our analysis identified two distinct resident macrophages with inflammatory and noninflammatory functions and characterized the successive stepwise development of T cell subtypes in the liver. Importantly, we uncovered possible molecular mechanisms and revealed the central regulation of macrophages on target cells of fatty liver by analyzing the cellular interaction between hepatocytes and immune cells. Our data provide valuable information for future research on NAFLD in zebrafish.

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“… 2012 ; Perli et al . 2020 ) except for VCF file intersection and annotation that was performed with VCFtools_0.1.13 (vcf-isec command) and SnpEff, respectively (Danecek et al . 2011 ; Cingolani et al .…”

Section: Methodsmentioning

confidence: 99%

Engineering of molybdenum-cofactor-dependent nitrate assimilation in Yarrowia lipolytica

Perli

Borodina

Daran

2021

FEMS Yeast Research

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Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y. lipolytica. The functionality of Moco was demonstrated by expression of an active Moco-dependent nitrate assimilation pathway from the same yeast donor, O. parapolymorpha. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate, reaching 0.13 h−1. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in S. cerevisiae demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts.

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Identification of oxygen-independent pathways for pyridine-nucleotide and Coenzyme-A synthesis in anaerobic fungi by expression of candidate genes in yeast

Cited by 3 publications

References 76 publications

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

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

Single-cell transcriptome landscape of zebrafish liver reveals hepatocytes and immune cell interactions in understanding nonalcoholic fatty liver disease

Engineering of molybdenum-cofactor-dependent nitrate assimilation in Yarrowia lipolytica

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