Molecular Regulation Mechanism Of High-Yield Laccase In Cerrena Sp.HYB07

Laccases are class of multi-copper oxidase,they have great potentials in various industrial applications with broad substrate specificity.However,the industrial application of laccase is limited by the low efficiency of laccase synthesis.Cerrena sp.strain HYB07,a white-rot fungus with high laccase yields had been bred in the laboratory.The efficient synthesis of laccase requires only copper ions and high carbon and nitrogen levels.We have analyzed the sequence,properties and transcripts of the eight laccases,and immobilized laccase applied to the decolorization of organic pollutants.In order to analyze the mechanism of transcriptional regulation of laccase on the molecular level,the genome and transcriptome sequences of Cerrena sp.HYB07 were obtained by high-throughput sequence platform.Five novel laccase genes were screened from the transcriptome data.The screened CUF1 transcription factor which was studied by Agrobacterium tumefaciens transformation technique.1.We have sequenced the genome of Cerrena sp.HYB07 and the result showed that total genome size is 44.2 megabases(Mb)with GC content of 50.65%,containing 8936 predicted genes.Fifteen laccases were screened and two of them were ferroxidase by Blast alignment,the other were laccase isozymes,according to the database annotation information.The strain showed low similarity by comparing genomics of Cerrena sp.HYB07 with Cerrena unicolor 303.2.Quantitative real-time polymerase chain reaction(qPCR)was used for transcription analysis of differentially expressed genes that cultured in different medium(Ys and Ys_LC,and Ys NoCu).In addition,a transcription factor CUF1 was screened from the transcriptome data.Its expression in Ys NoCu medium was 22.42 fold higher than that in Ys medium.3.According to the transcriptome data,5 novel laccase genes were cloned.The total 13 laccase isoenzymes including eight previous laccases(Lac1-8)constituted the laccase families of Cerrena sp.HYB07.The laccase families were analyzed intron characteristics,coding region sequence and characteristic sequence by bioinformatics software.4.The cuf1 gene and its cDNA sequence were cloned.The pCAMBIA1 302-cuf1 over-expression vector was transformed into Cerrena sp.HYB07 by Agrobacterium tumefaciens transformation technology.A pCAMBIA1302-cuf1 strain was obtained by screening and identification.The results showed that the activity of pCAMBIA1302-cuf1 strain was 22-54 fold higher than that of wild-type strain in Ys_NoCu medium.The activity of pCAMBIA1302-cuf1 strain is 0.7?2.7 fold higher than that of wild-type strain in Ys medium.Our study may pave the way for the further study of the transcriptional regulation mechanism of laccase.