Perturbations in the Heme and Siroheme Biosynthesis Pathways Causing Accumulation of Fluorescent Free Base Porphyrins and Auxotrophy in Ogataea Yeasts.

Azamat V Karginov,Vitaly V Kushnirov,Alexander I Alexandrov,Michael O Agaphonov

doi:10.3390/jof7100884

Abstract

The biosynthesis of cyclic tetrapyrrol chromophores such as heme, siroheme, and chlorophyll involves the formation of fluorescent porphyrin precursors or compounds, which become fluorescent after oxidation. To identify Ogataea polymorpha mutations affecting the final steps of heme or siroheme biosynthesis, we performed a search for clones with fluorescence characteristic of free base porphyrins. One of the obtained mutants was defective in the gene encoding a homologue of Saccharomyces cerevisiae Met8 responsible for the last two steps of siroheme synthesis. Same as the originally obtained mutation, the targeted inactivation of this gene in O. polymorpha and O. parapolymorpha led to increased porphyrin fluorescence and methionine auxotrophy. These features allow the easy isolation of Met8-defective mutants and can potentially be used to construct auxotrophic strains in various yeast species. Besides MET8, this approach also identified the HEM3 gene encoding porphobilinogen deaminase, whose increased dosage led to free base porphyrin accumulation.

Highlights

The biosynthesis of cyclic tetrapyrrol chromophores such as heme, siroheme, and chlorophyll involves the formation of fluorescent porphyrin precursors or compounds, which become fluorescent after oxidation
To identify mutations affecting synthesis of tetrapyrrole compounds in the methylotrophic yeast O. polymorpha, we performed a screen for mutants accumulating free base
Inactivation of the MET8 Gene in O. polymorpha and O. parapolymorpha Confers Red Fluorescence Excited by ~400 nm Light

Summary

Introduction

The biosynthesis of cyclic tetrapyrrol chromophores such as heme, siroheme, and chlorophyll involves the formation of fluorescent porphyrin precursors or compounds, which become fluorescent after oxidation. Besides MET8, this approach identified the HEM3 gene encoding porphobilinogen deaminase, whose increased dosage led to free base porphyrin accumulation. Tetrapyrrole compounds such as heme, chlorophyll, cobalamin, etc., accomplish many essential functions in different organisms (for a review see [1]). Their synthesis begins with formation of aminolevulinic acid either from glutamine (in plants, archaea, and the vast majority of bacteria) or from glycine and succinyl-CoA (in animals, fungi, and the α-subclass of the photosynthetic purple bacteria) [2]. Some other intermediates can be non-enzymatically oxidized to form fluorescent porphyrins such as uroporphyrin and coproporphyrin [3]

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Conclusion