The Identification Of YlHOG1-controlled Downstream Gene In The Osmotic Stress Response HOG Pathway In The Yeast Yarrowia Lipolytica

Every species has its ideal habitat.In the face of the complex and changeable environment,such as osmotic pressure,PH,temperature,oxygen,carbon source,nitrogen source and other conditions,the fungus has evolved its own way of survival.The high osmolarity glycerol(HOG)pathway is such a highly conservative signaling pathway responding to osmotic stress.It mediates the transcriptional activation of gene expression in order to adapt to the osmotic stress.The study of HOG pathway is very important to reveal the mechanism of the cell to cope with the external environment pressure.-It is also significant for the control of certain pathogens and dominant contaminant species during infection.Yarrowia lipolytica is a yeast species distantly related to the budding yeast Saccharomyces cerevisiae.It differs from S.cerevisiae in that it prefers lipids and protein as the major carbon source whereas S.cerevisiae prefers sugar.Due to its bio-safety,Y.lipolytica can be used to produce metabolic products such as citric acid and xylitol as well as single cell protein and gene engineering products,and has wide applications in industrial fermentation.Because yeast cells are growing in high osmolarity environment with high concentration sugars or salt,it is very important to understand the mechanism by which Y.lipolytica tolerates hyperosmotic stress during industrial fermentation.The high osmolarity glycerol(HOG)pathway involved in osmotic stress response was first identified in S.cerevisiae.The mitogen-activated protein kinase(MAPK)Hog1 is the key signaling molecule in the pathway.It plays an important role in the relaying of upstream signals to downstream genes.There are two signal pathways Sln1 and Sho1 upstream of Hog1.Downstream Hog1,a number of genes are transcriptionally controlled by Hog1.Y.lipolytica has proteins homologous to HOG pathway elements in S.cerevisiae.In this study,we investigated the function of Y.lipolytica YlHog1 kinase in osmotic stress response and the downstream target genes controlled by YlHog1.Firstly,we examined the ability to tolerate hyperosmotic stress in the Y.lipolytica wild-type strain and Ylhogl ? mutant.We found that the wild-type strain of Y.lipolytica has strong tolerance to osmotic stress.Its growth was not affected by the presence of 1.2 M NaCl.It can grow in the presence of 2.0 M NaCl.YIHog1 plays an important role in osmotic stress response which is manifested by the sensitivity of Ylhog1? mutant to 0.7 M NaCl,indicating that YIHog1 is critical for osmotic stress response in Y.lipolytica.Next,we have investigated the function of several Y.lipolytica proteins homologous to S.cerevisiae HOG pathway elements.We have found that the two transcription factors YlMsn4 and YISko1 are not important for osmotic stress response.However,we are not certain about the function of two protein kinases YlSte11 and YlSte20,which presumably acts upstream of YlHog1,in osmotic stress response.This result suggests that the target genes controlled by YlHog1 may not be the same as those controlled by S.cerevisiae Hog1.In addition,we found that similar to the protein kinase Sch9,which plays a role in osmotic stress response in S.cerevisiae,the Y.lipolytica YISch9 is also functionally important for osmotic stress response.Lastly,we performed a genetic screen to isolate genes that when overexpressed could restore tolerance to hyperosmotic stress to the Ylhog1 A mutant,aiming to find the target genes downstream of YlHog1.By random insertion of the strong hp4d promoter in different chromosomal locations in the Ylhog1? mutant,we have isolated 93 candidate transformants which showed enhanced tolerance to high NaCl concentration compared to the Ylhog1? mutant.The integration sites of hp4d on the chromosome of four candidates were determined by TAIL-PCR.In three candidates,there is a gene not far downstream of the hp4d integration site and the gene is in the same transcriptional direction as hp4d does.The three genes are YlCAC2,YlNCE103 and YIURE2D.They are thought to be overexpressed driven by the strong hp4d promoter in the cells.Unfortunately,later study showed that the three genes were not able to enhance osmotic stress tolerance in the Ylhogl? mutant.Our attempt to find YlHogl's downstream target genes did not succeed.Because currently there lacks reports on the mechanism of osmotic stress response in Y.lipolytica,this study has initiated the investigation.We believe that these investigations will reveal the difference in the mechanisms of osmotic stress response between Y.lipolytica and S.cerevisiae and will provide new insight in the understanding of how fungi tolerate osmotic stress.