Abstract
Methylotrophic yeast Pichia pastoris has proved to be especially useful for production of various heterologous proteins. In biotechnology it is very important to maintain the balance between high levels of heterologous gene expression and cell viability. Decisive understanding of gene regulation mechanisms is essential for reaching this goal. In this study, we investigated the effect of different nitrogen sources and phosphate concentration in media on methanol utilization. It was shown that expression levels of main genes, which are involved in methanol utilization (MUT genes) and in functioning of peroxisomes (PEX genes), are maximal when ammonium sulphate is used as a nitrogen source. Expression of these genes is decreased in media with poor nitrogen sources, such as proline. Addition of rapamycin to the media completely removed repression of AOX1 promoter in media with proline, which allows proposing that Tor-kinase is involved in establishing of nitrogen regulation of this gene. It was also shown that MUT genes expression levels get higher, when the phosphate concentration in media is increased.
Highlights
Microorganisms adaptation to different environment changes is primarily established on transcriptional level
In previous study it was found that type of nitrogen source presented in the medium influences the expression levels of AOX and PpCAT1 genes, which encode enzymes involved in first steps of methanol utilization [10]
Real-time PCR, which enables a quantitative analysis of gene expression levels, was used to investigate the effect of the nitrogen source and phosphate concentration on expression of chosen MUT genes
Summary
Microorganisms adaptation to different environment changes is primarily established on transcriptional level. The expression of most enzyme encoding genes changes dramatically in nutrient deficiency conditions. Yeast Pichia pastoris represent the eukaryotic group capable of utilizing methanol as a sole carbon source. Methanol utilization pathway is common in all methylotrophic yeasts and involves several unique enzymes [1]. Alcohol oxidase (Aox EC 1.1.3.13) catalyzes methanol oxidation to formaldehyde. Hydrogen peroxide, which is produced in this reaction, is degraded by catalase (Cat EC 1.11.1.6) to oxygen and water. Hydrogen peroxide formation is very dangerous for the living cell; alcohol oxidase and catalase are sequestered within special organelles—peroxisomes [2]. Proteins involved in peroxisome biogenesis are called peroxines (or PEX proteins) and are encoded by PEX genes [3]
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