Mycobacterium Neoaurum MN2 And MN4 Substrate And Product Tolerance Comparative Genomic Analysis

Androst-4-ene-3,17-dione(AD),as an important raw material and key intermediate in the synthesis of steroid hormones,almost all of the steroid hormone drugs used it as the starting material,was an indispensable drug in clinical.At present,in the world,AD as raw materials to produce a variety of pharmaceutical products which bring the gospel to the majority of patients in the world.Due to its mild reaction conditions,short cycle,high selection,high efficiency and environmental friendly features,microbial conversion method has been increasingly used in the production of steroid drugs.In this study,strians which we used are Mycobacterium neoaurum MN4 and Mycobacterium neoaurum MN2.The Mycobacterium neoaurum MN4 mutant strain can produce AD in high yield and can tolerate a higher concentration of the substrate phytosterol than the parent strain M.neoaurum MN2.Whole-genome sequence and transcriptome of the related strains were obtained by Illumina Solexa sequencing to study the high yield and substrate resistance mechanism of Mycobacterium neoaurum MN4.All these works may help us further study the genetic background information of two strains,the mechanism of sterol degrade into AD substrate and product tolerance and lay good foundation for molecular breeding.The results of this study are as follows:1.The genome sequencing and functional annotation of Mycobacterium neoaurum MN2 and MN4Whole-genome sequencing of MN2 and MN4 were carried out by Illumina Hiseq 2000 to produce 14 Mb and 8.1Mb raw data.The genome was assembled using pairedend reads by Velvet software,producing 43 and 33 contigs.The genome sequence was submitted to the NCBI database with accession numbers JXYZ00000000 and LQMX00000000,respectively.The size of Mycobacterium neoaurum MN2 was 5,390,529 bp,5,421,27 bp,with the GC content was 66.9%,containing 4890 CDSs,6 rRNA operons and 46 tRNAs.The size of Mycobacterium neoaurum MN2 was 5,421,27 bp,with the GC content was 66.9%,containing 4984 CDSs,6 rRNA operons and 46 tRNAs.2.Analysis of Mycobacterium neoaurum MN2 and Mycobacterium neoaurum MN4genomeThe main analysis content includes: COG and GO functional classification,basic metabolic analysis,Secondary metabolite analysis,comparative genomic analysis and gene analysis of Phytosterol degradation.All protein sequencs BLAST with COG and InterPro,then sorted them by function.The number of genes in metabolism,transcription,biosynthesis category is especially large,maybe means that both strains active.The metabolic pathways of in the both strains were reconstructed by the KEGG Pathway databaseand the sterol degradation pathway was analyzed.By mapping the reads for M.neoaurum MN4 to the genome of M.neoaurum MN2(using GATK and SAMTools),differences were identified and comprised one point INS,two point deletions and 181 SNPs variants.ChOs are flavoenzymes that catalyse the oxidation of sterols to sterones in the first step of steroid nucleus oxidation which was identified as a missense variant and FadE familly,Fad B familly,and FadA family also had missense.3.The comparative transcriptome analysis of Mycobacterium neoaurum MN2 and MN4The comparative analysis of transcriptome mainly including the calculation of difference in expression,the different expressed genes were enriched by GO and KEGG.Using the Bowtie2 to mapping the clean data to the reference gene,the difference was expressed by cufflink software.A total of 51 different genes were found,including 41 up-regulated genes and 10 down-regulated genes which found only one sterol degradation pathway gene CYP125A1 up-regulated,these differentially expressed genes were significantly enriched in Glucose metabolism,lipid metabolism,amino acid metabolism,energy metabolism and metabolic pathways of xenobiotics.In this study,the whole-genome and the comparative transcriptome of the AD producer MN2 and MN4 were studies.The ORF prediction,function annotation and classification analysis,comparative genome analysis were carried by some useful tools.the mutation site of MN4?the differentially expression gene between MN4 and MN2?the related genes and metabolic pathways of MN4 degradation of phytosterol metabolism were analyzed.These works lay a good foundation for molecular breeding study of AD producing bacteria.