Zeitschrift für analytische und bioanalytische Techniken

Abstrakt

Engineering Methylotrophic Yeasts for Biotechnology Applications

Dr. Hann Clara

Background: Because some yeast has evolved a methylotrophic lifestyle, they can use the single-carbon molecule methanol as a source of carbon and energy. Pichia pastoris (also known as Komagataella sp.) is one of them and is commonly employed for the generation of heterologous proteins as well as a model organism for organelle research. Our present understanding of the methylotrophic lifestyle is primarily based on extensive biochemical investigations that discovered numerous important methanol utilisation enzymes and their localization to the peroxisomes, including alcohol oxidase and dihydroxyacetone synthase. The pentose phosphate pathway is thought to be involved in C1 assimilation, but the specifics of these events are not yet understood.

Results: In this study, we compared the development of P. pastoris on a medium containing equal amounts of methanol and glycerol and glucose, as well as the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 processes. We discovered that the whole methanol absorption mechanism is restricted to peroxisomes as opposed to using a portion of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration, as was previously thought. P. pastoris (and perhaps other methylotrophic yeasts) have developed a duplicated set of methanol-inducible enzymes that are specific to peroxisomes for this purpose. Sedoheptulose-1,7- bisphosphate is used as an intermediary in this compartmentalised cyclic C1 assimilation mechanism known as the xylose-monophosphate cycle. The high demand for their respective cofactors, riboflavin, thiamine, nicotinamide, and heme, caused by the strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase, is reflected in the strong up-regulation of the corresponding synthesis pathways on methanol. Because of the high outflow towards methanol metabolic enzymes and their cofactors, methanol-grown cells contain more protein but fewer free amino acids. This illustrates an enhanced flow towards amino acid and protein synthesis and is also reflected in increased amounts of transcripts and/or proteins relevant to ribosome biogenesis and translation when taken in conjunction with up-regulation of several amino acid biosynthesis genes or proteins.

Conclusions: When taken as a whole, our study demonstrates how coordinated analysis of data from different systems biology levels can help reveal as-yet-unknown cellular pathways and completely change how we think about cellular biology.