| High osmolarity glycerol mitogen-activated protein kinase?HOG-MAPK?-mediated signaling transduction pathway,a highly conserved intracellular signal transduction pathway in eukaryotes,has been proved to be vital in stress responses including oxidative,organic acid,low pH,heat shock and low temperature stresses.However,the association of the HOG-MAPK pathway with the biosynthesis of polyunsaturated fatty acid?PUFA?at low temperature in oleaginous fungi has not been reported.Our previous studies have showed that overexpression of RKHog1 gene in the oleaginous yeast Rhodosporidium kratochvilovae YM25235could enhance the strain resistance to salt,osmolarity and low temperature stresses,and also increase the biosynthesis of linoleic acid?LA?and?-linolenic acid?ALA?in YM25235 at low temperature.These results indicated that the RKHog1 gene might be involved with the cold adaption of YM25235 strain and the biosynthesis increase of PUFA in YM25235 strain at low temperature.Therefore,in this study,the RKHog1gene in YM2535 will be knocked out by homologous recombination method,with the aim to further verify the function of RKHog1 gene in YM25235.Based on these,the relationship between RKHog1 and signal transduction pathway involved with the cold adaption of YM25235 and the biosynthesis of PUFA in YM25235 strain at low temperature will be uncovered.Firstly,the RKHog1 gene in YM2535 was knocked out by homologous recombination.Two genomic DNA fragments A and B with sizes of 1480 bp and 1820bp including RKHog1 genomic sequence and its upstream and downstream DNA sequences were amplified,respectively,and further subcloned into restrictive sites at both flanking sides of hygromycin B expression cassette in plasmid pRH2034 to generate a homologous recombinant fragment for RKHog1 gene knockout,which was then transformed into the YM25235 strain for homologous recombination by electroporation.672 transformants were obtained by hygromycin B resistance screening.Three RKHog1 gene knockout strains,designated as YM25235RKHog1?,were successfully obtained by stress resistance screening and PCR verification of genomic DNA.Stress resistance analysis showed that RKHog1 gene knockout resulted in reduced glycerol biosynthesis in YM25235 and reduced tolerance to salt stress,osmotic stress and growth at low temperature.Moreover,RKHog1 gene knockout significantly decreased the transcription level of?12/15-fatty acid desaturase gene RKD12 at low temperature,a key enzyme-encoding gene in the biosynthesis of LA and ALA in YM25235,which further resulted in a significant decrease in the biosynthesis levels of PUFA.The contents of LA and ALA was decreased from 27.76%and 7.97%in the control strain to 23.15%and 6.02%in the knockout strain,respectively,on the contrary to the same analysis results of the RKHog1 gene overexpressing in YM25235.These results indicated that RKHog1 protein regulates the biosynthesis of PUFA in YM25235 at low temperature,thus promoting the cold adaptation of YM25235.Meanwhile,consistent with similar reports,the RKHog1protein is also associated with the cell wall and membrane response to pressure and the regulation of cyclin in the YM25235.Recombinant RKHog1 gene expression plasmid pRGRKHog1 containing G418resistance marker was further constructed and subsequently transformed into the knockout mutant strain YM25235RKHog1?for the recovered expression of RKHog1gene,and the resultant knockin strain YM25235RKHog1?/pRGRKHog1 was successfully obtained.Same tress resistance analysis showed that the glycerol biosynthesis level and the resistance to salt stress,osmotic stress and low temperature stress of the RKHog1 knockout mutant strain YM25235RKHog1?were restored after RKHog1 gene complementation,as well as the transcription level of RKD12 gene and the biosynthesis level of PUFA at low temperature.The contents of LA and ALA in YM25235RKHog1?/pRGRKHog1 were 28.79%and 8.59%,respectively.These results further demonstrated that RKHog1 gene is not only associated with the resistance of YM25235 to salt stress and osmotic stress but also with the biosynthesis increase of PUFA at low temperature conditions,the latter promotes the better adaptation of the YM25235 to low temperature.In addition,cell wall and cell membrane pressure phenotypic analysis showed that the RKHog1 gene was also involved in the cell membrane and cell wall pressure response and the regulation of cyclin of the YM25235 strain.Many studies have shown that regulation of Hog1 activity is associated with its phosphorylation level.The analysis of RKHog1 protein phosphorylation level in the above-mentioned strains at 15°C showed that the RKHog1 gene-overexpressed strain YM25235/pRHRKHog1 exhibited the highest phosphorylation level,but no RKHog1protein phosphorylation was detected in the RKHog1 gene knockout mutant strain YM25235RKHog1?,whereas the RKHog1 protein phosphorylation in the RKHog1gene knockin strain YM25235RKHog1?/pRGRKHog1 was restored in comparison with that in the control strain.These data were consistent with the above-mentioned functional analysis results of these strains,indicating that low temperature promoted the phosphorylation level of RKHog1 protein and increased PUFA biosynthesis level,thus promoting the cold adaptation of YM25235.In addition,our previous studies have shown that histidine kinase HisK2301 and zinc finger transcription factor RKMsn4 might be involved with the biosynthesis increase of PUFA in YM25235 and cold adaptation of YM25235.In this study,RKHog1 gene knockout caused a significantly decrease in mRNA transcription levels of HisK2301 and RKMsn4 genes,indicating that,similar to HisK2301 and RKMsn4,RKHog1 is also involved in the signal transduction pathway which sense and transduce the low temperature signal inward and lead to the increase of PUFA biosynthesis.Furthermore,yeast two-hybrid analysis showed that RKHog1 interacted with RKMsn4,but the relationship between RKHog1 and HisK2301 remains to be further uncovered.Many studies have also shown that carotenoids are involved with microbial adaptation to environmental stresses.R.kratochvilovae YM2535 can synthesize carotenoids,such as torularhodin,torulin and?-carotene.Therefore,this work also analyzed the effect of RKHog1 gene knockout on the biosynthesis of carotenoid in YM25235.The results showed that the carotenoid content in YM25235RKHog1?was lower than that of the control strain cultured at both 30°C and 15°C,but more obvious decrease?33%of decrease?was observed at 15°C,indicating that the RKHog1 gene might also involve with carotenoid synthesis in YM25235 at low temperature.The mRNA transcriptional level of four carotenoid synthesis-related genes,3-hydroxy-3-methyl glutaric acid formyl CoA reductase gene RKHCR,3-hydroxy-3-methyl glutaric acid formyl CoA synthetase gene RKHCS,phytoene dehydrogenase gene RKPdH and Lycopene cyclase gene RKLC in YM25235 strain,was further analyzed.The results showed that the RKHog1 gene knockout caused a significant down-regulation of mRNA transcription of RKHCS gene,which was contrary to the increase of mRNA transcription level in the control strain at low temperature.These results indicated that RKHog1 is involved in biosynthesis regulation of carotenoids in YM25235 at low temperature,and RKHCS gene also plays an important role during this process.In conclusion,this study finally demonstrated that RKHog1 gene is not only involved with the resistance of YM25235 to salt stress and osmotic stress but also with biosynthesis regulation of PUFA at low temperature and cold adaptation of YM25235 by gene knockout and knockin strategies.And this involvement are also correlated with the HisK2301 gene and RKMsn4 gene revealed in our previously work.RKHog1 gene knockout caused a significantly decrease in mRNA transcription levels of HisK2301 and RKMsn4 genes,and RKHog1 interacted with RKMsn4,but the relationship between RKHog1 and HisK2301 remains to be further uncovered.Furthermore,RKHog1 could regulate the transcription level of RKHCS gene at low temperature,affecting the biosynthesis of carotenoid in YM25235.This study will contribute to elucidate the relationship between cold adaptation of YM25235 and the RKHog1-mediated signal transduction pathway responsible for the PUFA biosynthesis in YM25235 at low temperature,as well as the relationship between RKHog1 gene and the carotenoid biosynthesis at low temperature,providing basis for the fully uncovering of regulation mechanisms of PUFA and carotenoid biosynthesis at low temperature and for the application studies of industrial production of PUFA and carotenoid by R.kratochvilovae YM25235. |
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