Genome Sequencing And Comparative Analysis Of Industrially Relevant Aspergillus Strains

The development of massively high-throughput DNA sequencing technologiesthoroughly changed the study mode of classic genetics. Studies on the distributedgenetic markers was changed to in-depth analysis of whole genomic information.In-depth sequencing could contribute to understanding the genetic background andproducity of important industrial microbiological strains, and providing rich geneticinformation to analyze the potential of important industrial strains and improve theirproducing capacity.Filamentous fungi are one of the most important cell factories in the field ofindustrial biotechnology, including many GRAS certified strains. Aspergillus nigerSH2and Aspergillus oryzae AS3.863, used in this study, are two representatives. A.niger SH2is usually used to produce glucoamylase, whose distinct characteristics ishigh density fermentation without producing spores. A. oryzae AS3.863was isolatedfrom the natural koji of FUJIAN YONGCHUN, which owned high hydrolyzingactivity, strong growth capacity and strong resistance to environmental stress andother microorganisms. Its derivative strains play important roles in soy saucefermentation in China.In this study, the genome of A. niger SH2and A. oryzae AS3.863was sequencedby high-throughput Illumina sequencing technology, and subsequently assembled andannotated, using the genome of A. niger CBS513.88and A. oryzae RIB40as thereference genome. Comparative genomics was done to analyze the distinct industrialproducing capacity. Additionally, the genetic polymorphysm, systematic evolutionand codon bias of highly expressed genes were analyzed. The main content andconclusions in this study were as follows.1．whole genome sequencing and functional annotation of A. niger SH2and A. oryzaeAS3.86310.2Gb and2.6Gb reads data were obtained for A. niger SH2and A. oryzaeAS3.863using high-throughput Illumina sequcing technology, respectively, with theaverage sequencing depth of125.5fold for A. niger SH2and39fold for A. oryzae AS3.863. The genome was assembled using23.69M and7.8M paired-end reads byvelvet software, producing349contigs for A. niger SH2and921contigs for A. oryzaeAS3.863. The assembled genome sequence has been submitted to NCBI genomedatabase, with the accession no. AUZU00000000and PRJNA138081. The size of A.niger SH2genome is34.63Mb with GC content of50.26%, containing11517ORFsand267tRNAs. The size of A. oryzae3.863genome is36.41Mb with GC content of48.3%, containing11437ORFs and261tRNAs.2. Comparative genomic study between A. niger SH2and A. niger CBS513.88We constructed a method for syntenic analysis, SNP and indel analysis for A.niger SH2and A. niger CBS513.88. The approximately200kb deletion fragment inSH2strain was confirmed by PCR amplification. We found A. niger SH2lacked thegene related with the initiation of asexual sporulation (PrpA) which was located in the200kb deletion fragment, leading to its distinct aconidial phenotype. Frame shiftmutations and non-synonymous SNPs in genes of cell wall integrity signaling,β-1,3-glucan synthesis and chitin synthesis influence its cell wall development whichis important for its hyphal fragmentation during industrial high-efficiency proteinproduction.3. Comparative genomic study between A. oryzae AS3.863and A. oryzae RIB40Comparative analysis of A. oryzae AS3.863was done using A. oryzae RIB40asthe reference genome, including SNP analysis, selection pressure analysis and so on.We found certain genes of A. oryzae AS3.863with altered function due to genevariation. Gene function was analyzed based on their industrial application, whichcould provide a clue to genetic strain improvement.4. Phylogenetic analysis of A. niger strains and A. oryzae strainsPhylogenetic analysis showed the close relationship between A.niger SH2andA.niger CBS513.88, accordant with the result of syntenic analysis in this work. Wealso illustrated the relationship between A.oryzae and A. flavus strains. The obtainedresults provided a theoretical basis to determine the evolution status of A. niger SH2and A. oryzae AS3.863and their relevant strains.5. Codon usage bias for highly expressed genes in A. niger SH2and A. oryzae AS3.863Codons with high usage frequency in highly expressed genes of A. niger SH2and A. oryzae AS3.863were obtained by codon usage frequency analysis. The maindifference in codon usage between A. niger SH2and A. oryzae AS3.863exhibited inthe codon “CGT”,“CGC”,“AGC” and “GCT”. Codon bias analysis could contributeto the codon optimization and strain improvement for strains used in heterologousrecombinant protein expression.The aim of this study is to perform genome sequencing and annotation forindustrially relevant A. niger SH2and A. oryzae AS3.863using high-throughputsequencing technology. Based on the genome functional annotation, comparativegenomic analysis was conducted including their protein expression, phenotype variety,propagation, genetic evolution, physiology and biochemistry and so on, in order toprovide theoretical fundation for their application in industrial biotechnology.