The Structure And Functional Verification Of Recombinant Acyl-CoA Binding Protein From Zygosaccharomyces Rouxii

Acyl CoA binding protein(ACBP)is an approximately ~10 kDa protein and belongs to the conservative protein family which have been detected in all eukaryotic species.ACBP binds medium and long chain acyl-CoA esters with high specificity and affinity,which can be regulate the ?-oxidation of fatty acids,the elongate the fatty acid chains,and synthesize the sphingolipids.Zygosaccharomyces rouxii(Zr)is mainly used in the late fermentation of soy sauce,which can be survival in the fermentation with a salt content of 18% and can be improve the flavor of soy sauce.Most of the flavor compounds are esters,thus,it's of certain value to explore whether the related genes of fatty acid metabolism in Zygosaccharomyces rouxii have influence on the flavor substances.In this study,the function and structure of ACBP from Zygosaccharomyces rouxii were studied which lays a solid foundation for the follow-up research.The main research contents including:1.Cloning of Zracbp gene: there is one copy of Zracbp from Zygosaccharomyces rouxii.The Zracbp gene contains 264 bp base and encodes 87 amino acids with a molecular weight of about 10 kDa.Like most species of ACBP,ZrACBP does not contain transmembrane segments and signal peptides,which are intracellular proteins.Evolutionary analysis was showed high homology with saccharomyces cerevisiae.Analysis the ZrACBP structure which has high instability coefficient(>40)that's suggested the ZrACBP protein is unstable.Besides,ZrACBP contains a large hydrophobic fragment and has high lipid solubility coefficient.The secondary structure of ZrACBP is dominated by alpha helix followed by random curl,which is consistent with ACBP protein structures of most species.2.Expression,purification and functional identification of the recombinant ZrACBP-MBP protein: The prokaryotic over-expression vector pMAL-MBP-Zracbp have been constructed and transferred into Rosetta DE3 cells for inducing expression of recombinant protein.And then,purified the recombinant protein by amylose resin chromatography column and functional identified by MST.The results showed thatthe ZrACBP protein cloned into the pMAL-c4 X vector can be express in a large amount of recombinant ZrACBP-MBP protein.The quantitative purified protein was used to bind with nine Acyl-CoA esters.The experimental results showed that it was able to bind acyl-CoA esters,especially the medium and long-chain Acyl-CoA esters.The best affinity was C14:0-CoA.however,the binding capacity is one order of magnitude smaller than that of Saccharomyces cerevisiae,which suggested that it is possible the ZrACBP bind with Acyl-CoA is only a fraction of its function and may be involve in other metabolism.3.Analysing the structure of ZrACBP: The over-expression vector(pET-His-Zracbp)had been constructed,and the His-ZrACBP recombinant protein had been induced,purified,and concentrated.The recombinant protein had been used to protein crystallization,and the crystal had been diffracted for resolving the structure of ZrACBP.The results revealed that the crystals of ABCP belongs to space group P21 with cell parameters of a= 36.90 ?,b= 30.85 ?,c= 37.93 ?,?= 90.0,?=99.93,?=90.0.The Molecular replacement method was used to resolve the structure of ZrACBP under the 1.8 ? resolution,the results showed that ZrACBP was also composed of four helices bundles that form the basic bone of protein.The helix A1 from Q4 to A13,helix A2 from T22 to T37,helix A3 from I47 to E60,and helix A4 from K67 to Q87,respectively.The helices A1 and A2,helices A3 and A4 were connected by a random curl,the helices A2 and A3 not only connected by a random curl but also connected by ? structure.4.Analysing eukaryotic expression pattern of ZrACBP.In YPD medium,18%salt was added for simulating the high-salt fermentation environment of soy sauce.The results showed that the growth cycle of Zygosaccharomyces rouxii became longer,especially the subsequent fermentation time.The qRT-PCR method was used to determine the Zracbp expression of different growth stages.The results revealed that the expression of Zracbp was correlated with the growth curve.In the subsequent fermentation time,the increased expression of Zracbp may be related to the production of subsequent aroma substances.The over-expression Zracbp gene in yeast was not significant by transcriptome sequencing,which needed to be further optimized the experimental conditions.The uracil auxotrophic strains have beenconstructed which laids a certain foundation for the subsequent studies of Zygosaccharomyces rouxii.