Many organisms, including yeasts, obtain carbon and energy from carbohydrates, and nitrogen from other nutrients like amino acids. But some types of yeasts, like Pichia pastoris (P. pastoris), can use amino acids not only as a source of nitrogen, but also of carbon, finds a new study at the Indian Institute of Science (IISc), Bengaluru. The team, consisting of Umakant Sahu and Professor P N Rangarajan from the Department of Biochemistry, have studied the mechanism behind how P. pastoris can grow in a medium that does not contain glucose but only amino acids like glutamate, aspartate and proline.
Enzymes are biological catalysts that act on complex compounds and break them down into simpler molecules. They act on the food that we eat and help break it down to simple molecules so that our body can absorb and obtain nutrition and energy from food. These enzymes are manufactured in the body using the information coded in our genes. The genes that produce the enzymes are switched ‘on’ or ‘off’ as and when a nutrient becomes available in the medium. These switching of the genes are brought about by proteins called transcription factors in a process called ‘gene regulation’. The transcription factors bind to specific sequence elements in the promoter regions of these genes, the locations from where gene transcription usually begins.
Key regulator The researchers observed that a wild strain of yeast P. pastoris could grow in a glucose deficient medium containing only amino acids, whereas a mutant strain with a mutated gene that could not produce a protein Mxr1p, could not. This led them to believe that the protein Mxr1p was necessary to convert amino acids into carbon and nitrogen, acting as a transcription factor in P. pastoris. It was also found that Mxr1p acts as a key regulator for various genes like glutamate dehydrogenase, aspartate aminotransferases, malate dehydrogenase and glutamine synthetase, all of which are involved in the utilisation of amino acids as sole source of carbon.
During their experiments, the researchers observed that a mutant for glutamate dehydrogenase enzyme, ∆gdh2, was unable to grow in glucose deficient medium containing only amino acids. But, when a plasmid (extra chromosomal DNA) containing the GDH2 gene was introduced into these mutants, they were able to grow successfully in the same medium. This indicated that some enzyme is required to use amino acids as a source of carbon.
Mxr1p, the protein, regulates the transcription of the target genes by binding to a kind of sequence elements present in the promoters of these genes. In the course of their investigation, the researchers found that in a medium deficient in glucose, Mxr1p protein localises to the nucleus where the genes are present, proving that its transcription factor activity has been called for, under these circumstances.
Another interesting observation of the study was the regulation of some of the above genes in the ‘post transcriptional level’. Most genes are regulated, or switched on, in the initial stages of transcription where mRNAs are produced. But in some cases, genes can be regulated at a later stage during translation where proteins are produced from these mRNAs.
The researchers found that the protein Mxr1p regulated the production of glutamate dehydrogenase (GDH2), after transcription. The GDH2 mRNA levels in both the wild strain and the mutant strain (without the protein Mxr1p) of P. pastoris, grown in the same medium, were high and comparable. But the levels of the regulator protein gdh2p in these mRNAs were low in the mutant strain.
This shows that Mxr1p protein must be regulating the expression of GDH2 gene after the mRNAs were transcribed and before they were translated. It was also found that the ‘tail’ portion of the protein was involved in the inhibition of translation in gdh2p mRNAs. The conventional gene regulation activity by binding to the promoters was taken up by the ‘head’ portion of the protein as it contained a DNA binding domain. The researchers also rule out the possibility of Mxr1p directly binding to the RNA to inhibit translation since it localizes to the nucleus and is not available in the cytoplasm where translation happens. The researchers hypothesise that Mxr1p might be involved in the expression of some other gene whose product is required for the efficient translation of gdh2 mRNAs.
Insights P. pastoris is widely used as a host cell for the production of proteins in the lab since it grows high in a dense manner and gives a very good protein yield. Being respiratory yeast, P. pastoris completely oxidises sugars, avoiding the formation of ethanol. As a result, it efficiently utilises the carbon sources and yields a high biomass.
Amino acids are routinely added to the growth medium along with other conventional carbon sources. They enhance growth rate and recombinant protein production in which amino acids primarily serve as precursors for the synthesis of proteins. During high cell density fermentation of P. pastoris, ammonium sulphate or organic nitrogen sources such as amino acids are used as nitrogen sources.
More studies based on these results may provide new insights into potential interactions between metabolism and amino acid metabolism on recombinant protein production, which may lead to novel biotechnological applications.
(The author is with Gubbi Labs, a Bengaluru-based research collective)