Identification of Oxygen-Independent Pathways for Pyridine Nucleotide and Coenzyme A Synthesis in Anaerobic Fungi by Expression of Candidate Genes in Yeast

Abstract

Neocallimastigomycetes are unique examples of strictly anaerobic eukaryotes. This study investigates how these anaerobic fungi bypass reactions involved in synthesis of pyridine nucleotide cofactors and coenzyme A that, in canonical fungal pathways, require molecular oxygen. Analysis of Neocallimastigomycetes proteomes identified a candidate L-aspartate-decarboxylase (AdcA) and L-aspartate oxidase (NadB) and quinolinate synthase (NadA), constituting putative oxygen-independent bypasses for coenzyme A synthesis and pyridine nucleotide cofactor synthesis. The corresponding gene sequences indicated acquisition by ancient horizontal gene transfer (HGT) events involving bacterial donors. To test whether these enzymes suffice to bypass corresponding oxygen-requiring reactions, they were introduced into fms1∆ and bna2∆ Saccharomyces cerevisiae strains. Expression of nadA and nadB from Piromyces finnis and adcA from Neocallimastix californiae conferred cofactor prototrophy under aerobic and anaerobic conditions. This study simulates how HGT can drive eukaryotic adaptation to anaerobiosis and provides a basis for elimination of auxotrophic requirements in anaerobic industrial applications of yeasts and fungi. IMPORTANCE NAD (NAD
⁺) and coenzyme A (CoA) are central metabolic cofactors whose canonical biosynthesis pathways in fungi require oxygen. Anaerobic gut fungi of the Neocallimastigomycota phylum are unique eukaryotic organisms that adapted to anoxic environments. Analysis of Neocallimastigomycota genomes revealed that these fungi might have developed oxygen-independent biosynthetic pathways for NAD
⁺ and CoA biosynthesis, likely acquired through horizontal gene transfer (HGT) from prokaryotic donors. We confirmed functionality of these putative pathways under anaerobic conditions by heterologous expression in the yeast Saccharomyces cerevisiae. This approach, combined with sequence comparison, offers experimental insight on whether HGT events were required and/or sufficient for acquiring new traits. Moreover, our results demonstrate an engineering strategy for enabling S. cerevisiae to grow anaerobically in the absence of the precursor molecules pantothenate and nicotinate, thereby contributing to alleviate oxygen requirements and to move closer to prototrophic anaerobic growth of this industrially relevant yeast.