| Microbial fatty acid as raw material for biodiesel has good prospects,however,separation and purification processes become important bottlenecks for the purity and costs of fatty acid.Exploration of the extracellular secretion mechanisms of fatty acid is basic scientific questions for microbial cell transformation,promoting microbial fatty acid convert green energies such as biodiesel fuel.The study using proteomics technologies(two-dimensional gel electrophoresis)and transcriptomics technologies(RNA-Seq)analysed the different expression of proteins and mRNAs in different periods of fatty acid secretion between Saccharomyces cerevisiae knocking out acyl-CoA synthetases and Saccharomyces cerevisiae not knocking out acyl-CoA synthetases.Besides,this study also analysised the effects of knocking out acyl-CoA synthetases on the global regulation,cellular metabolism and possible secretion mechanism;sumed up the hypothetical mechanisms of fatty acid extracellular secretion in Saccharomyces cerevisiae.The main research results were as follows:(1)Preparation of protein sample is critical for two-dimensional electrophoresis,and cell disruption,protein solubility,protein modification and degradation will directly affect the accuracy and repeatability of 2-DE.According to the protein release rate and the protein concentration,this study compared six.methods of protein preparation for Saccharomyces cerevisiae and the best method was glass beads breaking.Based on the glass beads breaking method reported previously,breaking times,dosage of glass beads and loading ratio of liquid were further optimized by single factor and orthogonal test.The optimal conditions for holoprotein preparation were 50 mg dry weight of Saccharomyces cerevisiae,1 mL lysis buffer,1g glass beads,20%loading ratio of liquid,vortexing 30 s,ice bath 30 s,repeating above operations 60 times.The optimal yield and concentration of protein were 9.23%and 1.21 g/L.(2)So to analysis the differences of protein and RNA expression in different cell growth periods and fatty acid secretion periods between mutation strain and original strain,the phenotype of fatty acid secretion of the two strains first need to verify.The extracellular and total fatty acid production of YB526 is higher than YB332,confirming that the fatty acid secretion type of the two strains is correct.The time of collecting fermentation liquor for exponential phase of YB332 is 26h and the time of collecting fermentation liquor for exponential and stationary phase of YB526 is 30h and 80h by fatty acid secretion map.(3)This study combining proteomics technologies and transcriptomics technologies analysised the different RNA and protein expression levels between the mutant and the original strains in the exponential phase and stationary phase.The deficiency of acyl-CoA synthetases in Saccharomyces cerevisiae resulted in a series of cellular responses in YB526 owing to the accumulation of fatty acid.Firstly,the amino acids including glutamate,arginine,lysine and ornithine were taken advantage by the acid resistance systems to reduce the toxic effects of fatty acid.Secondly,the content of ergosterol and ceramide was reduced in cell envelope,which improved the permeability of the membrane and strengthened the free diffusion of fatty acid out of cells.Thirdly,the increase of 14-3-3 proteins could not improve the biosynthesis of ergosterol in the acyl-CoA deficiency strain YB526 due to the toxic of fatty acid.(4)Differential gene and protein expression showed that the membranes in mutants really had changed,concluding that the hypothetical mechanism of fatty acid extracellular secretion of Saccharomyces cerevisiae.Firstly,efflux pumps could discharge fatty acid from intracellular to the extracellular environment.Secondly,the decrease of ergosterol and ceramide in cell envelopes could improve permeability of membranes and the secretion of fatty acid. |
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