Cloning And Identification Of Acyl-coenzyme A Binding Protein Gene From Aspergillus Oryzae

Aspergillus oryzae has been used for production of tranditional soybean sauce and soy for over 1000 years in China,without any safety problems,and in fact A.oryzae has been granted GRAS status and it is also the ideal host of heterologous protein expression.Although the whole A.oryzae genome sequence was released in 2005,the functions of many important genes remain to be elucidated.The acyl-CoA binding protein gene of A.oryzae is one of such genes to be investigated.To clone and characterize the acyl-CoA binding protein gene in A.oryzae 3.024,the following research work has been carried out:1)A full-length cDNA encoding a candidate ACBP from A.oryzae(AoACBP)was cloned and expressed in E.scherichia coli Rosetta(DE3)cells as a maltose-binding protein(MBP)fusion protein.The MBP-AoACBP protein was purified by an amylose resin chromatography column.SDS-PAGE showed that MBP-AoACBP has an estimated molecular weight of 82 kDa;2)microscale thermophoresis binding assay showed that the recombinant AoACBP displayed much greater affinity for C20:0 acyl-coenzyme A(Kd=23 nM)than for other acyl-coenzyme A,thus demonstrating the preference of AoACBP for long-chain acyl-CoA and playing an important role in the process of long-chain acyl coenzyme A transporter;3)a uridine auxotrophic A.oryzae strain was obtained by UV mutagenesis as an expression host,expression vector ANEp8-acbp was constructed by inserting acyl-CoA binding protein gene into a motified ANEp8-1 vetor;4)To explore the potential function of the A.oryzae ACBP,AoACBP,in the fungal life cycle,its distribution pattern and expression levels from vegetative growth at 24 h to asexual spore production at 72 h was investigated by RT-PCR and Western blot analysis.A significant increase of AoACBP contents was detected between 60 h and 72 h by Western blot analysis,while the transcription level of acyl-coenzyme A-binding protein gene showed no significant variations in different growth stages.The above results have provided a foundation for elucidating the function of acbp gene and its applications.