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

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.

SIN-like pathway kinases regulate the end of mitosis in the methylotrophic yeast Ogataea polymorpha

The Mitotic exit network (MEN) is a conserved signalling pathway essential for termination of mitosis in the budding yeast Saccharomyces cerevisiae. All MEN components are highly conserved in the methylotrophic budding yeast Ogataea polymorpha, except for Cdc15 kinase. Amongst O. polymorpha protein kinases that have some similarity to ScCdc15, only two had no other obvious homologues in S. cerevisiae and these were named OpHCD1 and OpHCD2 for homologue candidate of ScCdc15. A search in other yeast species revealed that OpHcd2 has an armadillo type fold in the C-terminal region as found in SpCdc7 kinases of the fission yeast Schizosaccharomyces pombe, which are homologues of ScCdc15; while OpHcd1 is homologous to SpSid1 kinase, a component of the Septation Initiation Network (SIN) of S. pombe not present in the MEN. Since the deletion of either OpHCD1 or OpHCD2 resulted in lethality under standard growth conditions, conditional mutants were constructed by introducing an ATP analog sensitive mutation. For OpHCD2, we constructed and used new genetic tools for O. polymorpha that combined the Tet promoter and the improved auxin-degron systems. Conditional mutants for OpHCD1 and OpHCD2 exhibited significant delay in late anaphase and defective cell separation, suggesting that both genes have roles in mitotic exit and cytokinesis. These results suggest a SIN-like signalling pathway regulates termination of mitosis in O. polymorpha and that the loss of Sid1/Hcd1 kinase in the MEN occurred relatively recently during the evolution of budding yeast.

GENOMIC DIVERSITY, CHROMOSOMAL REARRANGEMENTS, AND INTERSPECIES HYBRIDIZATION IN THE OGATAEA POLYMORPHA SPECIES COMPLEX

The methylotrophic yeast Ogataea polymorpha has long been a useful system for recombinant protein production, as well as a model system for methanol metabolism, peroxisome biogenesis, thermotolerance, and nitrate assimilation. It has more recently become an important model for the evolution of mating-type switching. Here, we present a population genomics analysis of 47 isolates within the Ogataea polymorpha species complex, including representatives of the species O. polymorpha, O. parapolymorpha, O. haglerorum, and O. angusta. We found low levels of nucleotide sequence diversity within the O. polymorpha species complex and identified chromosomal rearrangements both within and between species. In addition, we found that one isolate is an interspecies hybrid between O. polymorpha and O. parapolymorpha and present evidence for loss of heterozygosity following hybridization.

Random Transfer of Ogataea polymorpha Genes into Saccharomyces cerevisiae Reveals a Complex Background of Heat Tolerance

Horizontal gene transfer, a process through which an organism acquires genes from other organisms, is a rare evolutionary event in yeasts. Artificial random gene transfer can emerge as a valuable tool in yeast bioengineering to investigate the background of complex phenotypes, such as heat tolerance. In this study, a cDNA library was constructed from the mRNA of a methylotrophic yeast, Ogataea polymorpha, and then introduced into Saccharomyces cerevisiae. Ogataea polymorpha was selected because it is one of the most heat-tolerant species among yeasts. Screening of S. cerevisiae populations expressing O. polymorpha genes at high temperatures identified 59 O. polymorpha genes that contribute to heat tolerance. Gene enrichment analysis indicated that certain S. cerevisiae functions, including protein synthesis, were highly temperature-sensitive. Additionally, the results confirmed that heat tolerance in yeast is a complex phenotype dependent on multiple quantitative loci. Random gene transfer would be a useful tool for future bioengineering studies on yeasts.

Genome-scale model reconstruction of the methylotrophic yeast Ogataea polymorpha

Background Ogataea polymorpha is a thermotolerant, methylotrophic yeast with significant industrial applications. While previously mainly used for protein synthesis, it also holds promise for producing platform chemicals. O. polymorpha has the distinct advantage of using methanol as a substrate, which could be potentially derived from carbon capture and utilization streams. Full development of the organism into a production strain and estimation of the metabolic capabilities require additional strain design, guided by metabolic modeling with a genome-scale metabolic model. However, to date, no genome-scale metabolic model is available for O. polymorpha. Results To overcome this limitation, we used a published reconstruction of the closely related yeast Komagataella phaffii as a reference and corrected reactions based on KEGG and MGOB annotation. Additionally, we conducted phenotype microarray experiments to test the suitability of 190 substrates as carbon sources. Over three-quarter of the substrate use was correctly reproduced by the model and 27 new substrates were added, that were not present in the K. phaffii reference model. Conclusion The developed genome-scale metabolic model of O. polymorpha will support the engineering of synthetic metabolic capabilities and enable the optimization of production processes, thereby supporting a sustainable future methanol economy.