Study On The Key Gene Of High Osmolality Glycerol Pathway-CgHOG1in Candida Glycerinogenes

Candida glycerinogenes, one of the most excellent glycerol-producer, has beencommercially exploited to large-scale glycerol production. However, our knowledge about themolecular level and genetic background of C. glycerinogenes lags far behind those of modelyeasts such as Saccharomyces cerevisiae. The high osmolarity glycerol mitogen-activatedprotein kinase (HOG-MAPK) pathway, mediated by Hog1, is one of the most important signaltransduction pathways in S. cerevisiae. However, little information about HOG-MAPKpathway is available in C. glycerinogenes In this study, a novel HOG1gene from C.glycerinogenes was cloned and its function was also analyzed in S. cerevisiae hog1Δ nullmutant strain.The complementation of HOG1gene in S. cerevisiae hog1null mutantdemonstrates that the gene obtained in this study was functional.A1164bp gene from C. glycerinogenes homologous to HOG1of other yeasts wasobtained using degenerate primers PCR in conjunction with Self-formed Adaptor PCR andnamed CgHOG1. Sequence analysis revealed that the open reading frame of CgHOG1encoding a putative peptide of387deduced amino acids. Sequences alignment revealed thatCgHOG1showed the highest identity to the HOG1of Naumovozyma castellii (81%) and76%to the S. cerevisiae HOG1gene. A catalytic protein kinase domain is from Tyr-24to Leu-302,which is similar to other MAPK (Mitogen-activated protein kinase) Hog1. Inside the catalyticdomain, there are an active site with Asp-144, a TGY motif, ATP-binding signature of proteinkinases and a common docking (CD) motif.Heterologous expression of CgHOG1gene in S. cerevisiae hog1null mutant showed anincrease in salt-tolerance and glycerol production. Functional analysis revealed that S.cerevisiae hog1null mutant transformed with CgHOG1was restored to the wild-typephenotype when cultured in high osmolarity media. After NaCl shock, the transcription ofGPD1in CgHOG1-harbouring strains was induced markedly, to the same level as the S.cerevisiae hog1wild-type W303-1A strain, but the S. cerevisiae hog1null mutant failed toenhance the transcription of GPD1under the same conditions. It is supposed that CgHogl canregulate the transcription of GPD1in S. cerevisiae and control the production of intracellularglycerol, thereby enhancing the resistance of cells against osmotic pressure.Further investigation of CgHOG1transcription levels under different glucose and NaClconcentrations revealed a smooth trend with increasing glucose and NaCl concentrations,while the transcriptional level of CgGPD revealed a flunctuant trend along with the yield ofintracellular glycerol. It was demonstrated that under certain hypertonic conditions (~300g/Lglucose), CgHog1could obviously regulate the transcription of CgGPD gene, and promotethe production of intracellular glycerol.