HOG Response Pathway And The Osmotic Stress Tolerance Of Candida Glycerinogenes

Candida glycerinogenes,the excellent glycerol producer with hyperosmotic tolerance and multi-stress resistance,is considered to be a potential biotechnological host.The high osmolarity glycerol?HOG?response pathway mediated by Hog1 is the most important mechanism with the challenge of environment stress,it plays a critical role when the cells are exposed to osmotic stress,oxidative stress,heat shock,organic acid and low pH.However,the study on HOG pathway of C.glycerinogenes is limited because of the lack of genetic tools.In this study,on the basis of whole genome sequencing,we constructed the genetic tools for the diploid industrial yeast to investigate Hog1,the core component of HOG pathway and the osmo-adaptation of C.glycerinogenes.The main results are highlighted as follows:?1?To obtain available genetic tools for the diploid industrial yeast C.glycerinogenes,an effective selection marker during molecular manipulation is required.In this study,a uracil auxotroph mutant was obtained by using the atmospheric and room temperature plasma?ARTP?method.The results of sequencing indicated that the orotate binding site in orotate phosphoribosyltransferase of U5 was mutate.U5 maintains the high tolerance to hyperosmotic,high temperature,and low pH,and it is used as subject in the future molecular manipulation.An integration expression vector pURGAP was constructed by using URA5 as selection marker.The positive rate of transformant was reached more than 85%.In addition,we constructed the gene knock-out vector pHUH,and the gene knock-out in diploid industrial yeast C.glycerinogenes was studied for the first time.?2?HOG pathway mediated by the core component Hog1 plays the most important role during the osmotic response.The homologous genes involved in HOG pathway were obtained via the analysis of genome sequencing.The results of bioinformatics analysis and functional complementarity experiment indicated that the signal transduction might only dependent on Sln1 branch.The function of core component Hog1 was studied by functional complementarity and western blotting analysis.The HOG1 null mutant showed extreme sensibility to osmotic stress,acetic acid and H₂O₂.Especially,the osmotic tolerance of C.glycerinogenes is highly dependent on Hog1 to campare with Saccharomyces cerevisiae.The pseudohypha generation of HOG1?under non-induced condition indicated that Hog1 has cross talk with the morphogenesis.The results above hightlighted the different HOG pathway in C.glycerinogenes.?3?Accumulation of glycerol under the control of HOG pathway is the major response mechanism in yeast to face the challenge of hyperosmotic stress.The homologous genes involved in glycerol metabolism and transport were obtained from the results of genome sequencing.The key enzyme gene of glycerol synthesis pathway GPD1 and glycerol uptake protein STL1 was identified as the target gene of Hog1 by using chromatin immunoprecipitation?ChIP?-PCR.Deletion of GPD1 eliminated the glycerol production,and affected the cell growth under high temperature and oxidative stress.Unlike S.cerevisiae,GPD1?showed serious growth defects under extreme osmotic stress,but maintained high tolerance to moderate osmotic stress.These results indicated that response mechanism except for glycerol synthesis might also exist in C.glycerinogenes to play an important role.?4?Results of ChIP-PCR in the previous study showed that the glycerol uptake protein gene STL1 is the target of Hog1.Exogenous addition of small amount of glycerol shortened the delayed cell growth and improved the final biomass of C.glycerinogenes under hyperosmotic stress.These results implies the occurrence of glycerol uptake under osmotic stress dependent on Hog1.Two homologous of glycerol uptake protein Stl1 and Stl2 were obtained on the basis of whole genome sequencing,however no homologous to the typical aquaglyceroporin Fps1can be found.Stl1 and Stl2 were identified as transmembrane protein by structure prediction and subcellular localization.The results of functional complementarity and gene knock-out highlighted their roles in glycerol uptake.Transcriptions of STL1 and STL2 under osmotic stress are different from each other.The expression of STL1 were osmotic-induced whereas that of STL2 was constitutive.Especially,the upregulation of STL1 extended to more than 120 min in the GPD1 deletion mutant,indicated a remedial way of glycerol uptake by Stl1.?5?To obtain the additional mechanism during the osmotic response,RNA-Seq was performed to study the whole transcriptome of C.glycerinogene exposed to osmotic stress.A total of 87 up-regulated and 180 down-regulated genes was identified as differentially expressed genes.Among them,PUT1,PUT2,ARG3 and ARG4 involved in arginine synthesis pathway were upregulated,and CAR1,CAR2 involved in arginine catabolic pathway were downregulated.The concentration of intracellular arginine increased by 183%in the presence of 1 M NaCl.The transcriptional regulation of related genes and accumulation of arginine did not occurred,highlighting the role of Hog1in the osmotic response.The results of RNA-Seq and qRT-PCR showed that the transcription of specific?-aminobutyric acid permease gene UGA4 was osmotic-induced depending on Hog1.Accumulation of?-aminobutyric acid was occurred under osmotic conditions when it is added exogenously and the intracellular?-aminobutyric acid contents were significantly enhanced with the increasing osmolarity.In addition,overexpression of the glutamate decarboxylase gene GAD1 resulted in an increased intracellular GABA and improvement in cell growth under hyperosmotic conditions.These results indicates that C.glycerinogene operates amino acid synthesis and transport by HOG pathway to respond the osmotic stress...