Effects Of HisK2301 Gene Deletion On The Adaptation Of Rhodosporidium Kratochvilovae YM25235 At Low Temperature

Two-component signal transduction system(TCS)is also widely distributed in eukaryotes.It plays an important role in the stress response of cells,especially in tolerance to high osmotic,oxidative,salt and low temperature stresses.Studies have shown that two-component signal transduction system(TCSTS)in protokaryotes could regulate the polyunsaturated fatty acids(PUFA)biosynthesis and promote the cell adaptation at low tempertarue,but similar mechanisms have not been reported in fungi.Our previous data have showed that overexpression of histidine kinase gene HisK2301 in an oleaginous red yeast strain—Rhodosporium kratochvilovae YM25235 with could significantly improve adaptation of YM25235 to salt and osmotic stresses,and also increase the biosynthesis of glycerol,linoleic acid(LA)and ?-linolenic acid(ALA)at low temperature and enhanced the cold adaptation of YM25235.This indicats that histidine kinase HisK2301 might be correlated with the cold adaptation of YM25235 and the regulation of PUFA biosynthesis at low temperature in YM25235,but its specific function needs further analysis and verification.In this study,HisK2301 gene in YM25235 was knocked out by homologous recombination.The resultant mutant strain YM25235Hisk2301? was successfully obtained by stress resistance screening and PCR verification of genomic DNA.Function analysis results showed that HisK2301 gene knockout significantly reduced tolerance of YM25235 to salt,osmotic,and oxidative stresses,as well as cold adaptation.Meanwhile,the biosynthesis of glycerol and PUFA in YM25235 was significantly decreased at low temperature,the content of PUFA(including LA and ALA)decreased from 31.85% and 9.56% in YM25235 to 25.51% and 5.67% in the mutant strain YM25235 HisK2301?,respectively.Based on this,the gene HisK2301 was further transformed back into YM25235 HisK2301?for reexperssion.The analysis results also showed that HisK2301 gene reexpression significantly restored the tolerance of the mutant strain to the above-mentioned stresses and the biosynthesis level of glycerol and PUFA at low temperature.These results proved again that HisK2301 gene is associated withonot only the tolerance of the YM25235 strain to salt,osmotic and oxidative stresses but also the regulation of biosynthesis of glycerol and PUFA at low temperature and the cold adaptation of YM25235 to low temperature.Changes in transcription levels of glycerol biosynthesis-related gene RKGPD1 and PUFA biosynthesis-related gene RKD12 at low temperature also conformed this association.In addition,analysis also showed that knockout of HisK2301 gene caused down-regulation of the transcription levels of RKHog1 and RKMsn4 genes at low temperature.Combined with our previous function analysis results of RKHog1 and RKMsn4 genes,we can infer that cold signal transduction pathway mediated by histidine kinase HisK2301 and regulation of glycerol,PUFA and carotenoid biosynthesis at low temperature were also involved with HOG-MAPK signaling pathway and zinc finger transcription factor RKMsn4,but their specific interactions remain to be further studied.Many studies have showed that the composition and content of carotenoid are related to the adaptation of microorganisms to various environmental stresses,such as low temperature,oxidative and strong light stresses.Our previous data also showed that the colonies of YM25235 in color was darker at low temperature.Therefore,effect of HisK2301 gene knockout on the carotenoid biosynthesis in YM25235 was further studied.The results showed that low temperature induced a increase of oxidative stress in YM25235,which led to the content of carotenoid increased significantly from 2.03mg/g DCW to 3.54mg/g DCW.However,gene knockout significantly inhibit this increase,but it was restored after HisK2301 gene reexpression.Moreover,analysis results also showed that increase of ?-carotene content was the most significant at low temperature among the four carotenoid components in YM25235.These results indicated that composition and content of carotenoids might also affect the cold adaptation of YM25235,and the histidine kinase HisK2301 is involved in the regulation of carotenoid biosynthesis in YM25235 at low temperature,which was also further confirmed by the transcriptional changes of carotenoid biosynthesis-related genes.In conclusion,this study proved that histidine kinase HisK2301 is involved in the tolerance of YM25235 to salt,osmotic and oxidative stresses.It is also responsible for the regulation of glycerol,PUFA and carotenoid biosynthesis in YM25235 at low temperature,affecting cold adaptation of YM25235.Low temperature signal transduction pathway mediated by histidine kinase HisK2301 and its regulation of glycerol,PUFA and carotenoid biosynthesis involve with HOG-MAPK signaling pathway and zinc finger transcription factor RKMsn4.This study is helpful to entirely illustrate the HisK2301-mediated signal transduction pathway and its potential regulatory mechanisms for the biosynthesis of glycerol,PUFA and carotenoid in YM25235,and lay a foundation for further uncovering the cold adapted mechanisms of YM25235 and related theoretical and applied research.