| During the fermentation process,the cell production capacity often leads to an increase in fermentation temperature,abnormal growth and metabolism,and cooling is required for normal fermentation,resulting in high energy consumption.Candida glycerinogenes,as an excellent industrial stress resistant strain,exhibits obvious growth advantages under high temperature compared with the model strain Saccharomyces cerevisiae,and has good tolerance to high temperature stress.Promoters are important components for gene expression regulation.Screening high temperature inducible promoters from C.glycerinogenes can provide a basis for the transformation of industrial yeast strains.The research content of this paper were as follows:According to the transcriptome data of C.glycerinogenes combined with the literature of S.cerevisiae high temperature stress related genes,thirty-three potential high temperature stress response genes were screened from C.glycerinogenes,and the genes under high temperature stress were further detected by fluorescent quantitative PCR technology.Relative transcription level,it was found that the transcription levels of CWP1,AAC,POT1,HSP12,ATP1,INO1 and SCL genes were most up-regulated under high temperature stress.The promoters corresponding to the cloned genes were analyzed by using the online analysis software JASPAR database to analyze the binding sites of high temperature related transcription factors in the promoter sequence,and it was found that the promoters all had characteristic sites for the regulation of high temperature transcription factors.Using the green fluorescent protein gene GFP as a reporter gene,a high temperature inducible promoters recombinant expression vectors were constructed,and transferred to C.glycerinogenes competent cells to obtain fluorescent detection recombinant strains,and the fluorescence intensity of the recombinant strains cultured at different temperatures were detected using a fluorescent microscope.The fluorescence intensity gradually increased with the increase of temperature,and the seven promoters were all high temperature induced promoters.In order to investigate the versatility of the high temperature inducible promoter,using the model strain S.cerevisiae as the host cell to construct a high temperature inducible promoters recombinant plasmids,which were transferred into S.cerevisiae competent cells to obtain fluorescent detection of recombinant strains,and detection of recombinant strains after cultivation at different temperatures fluorescence intensity,it was found that the fluorescence intensity gradually increased with increasing temperature.Therefore,the promoters PCgcwp1,PCgaac,PCgpot1,PCghsp12,PCgatp1,PCgino1 and PCgscl were all high temperature inducible promoters commonly used in yeast cells,of which PCgcwp1 was the strongest promoter.In order to study the industrial application of the promoter,the XYL1 gene was cloned,connected to the expression vector of the promoter,and the expression vector were transformed into C.glycerinogenes competent cells to construct seven xylitol producing recombinant strains,The seven recombinant had high enzyme activity at 42°C,and the recombinant enzyme C.g-PCgcwp1-xyl1 had the highest enzyme activity,which was 3.8times that of enzyme activity at 42°C.The fermentation results showed that the XYL1 gene was successfully expressed in C.glycerinogenes,and all seven recombinant strains could produce xylitol,and the xylitol production at 42°C was higher than that at 30°C.Among them,the recombinant strain C.g-PCgcwp1-xyl1 has the highest xylitol production,reaching 15.4 g·L-1,which was 204.3%higher than that at 30°C.It showed that the high temperature inducible promoters could enhance the expression of XYL1 gene at high temperature,and make the transformant had stronger xylitol synthesis ability,which can provided a reference for the molecular transformation of high temperature fermentation strains. |
PDF Full Text Request