Genome re-sequencing and reannotation of the Escherichia coli ER2566 strain and transcriptome sequencing under overexpression conditions
| Autor: | Zhibo Kong, Ying Gu, Jiajia Li, Shaowei Li, Yingbin Wang, Liqin Liu, Ningshao Xia, Yuqian Li, Qingbing Zheng, Lizhi Zhou, Hai Yu, Kaihang Wang, Yue Ma, Yang Huang, Tingting Chen |
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| Rok vydání: | 2020 |
| Předmět: |
RNA
Untranslated lcsh:QH426-470 lcsh:Biotechnology Pseudogene Bacterial genome size Computational biology Engineer bacteria Biology Genome 03 medical and health sciences symbols.namesake lcsh:TP248.13-248.65 Escherichia coli Genetics RefSeq Transcriptome sequencing Escherichia coli ER2566 Gene 030304 developmental biology Sanger sequencing 0303 health sciences Base Sequence Whole Genome Sequencing Escherichia coli Proteins 030302 biochemistry & molecular biology Molecular Sequence Annotation Gene Expression Regulation Bacterial lcsh:Genetics Gene nomenclature Lac Operon symbols DNA microarray Transcriptome Genome reannotation Genome Bacterial Research Article Biotechnology |
| Zdroj: | BMC Genomics BMC Genomics, Vol 21, Iss 1, Pp 1-11 (2020) |
| DOI: | 10.21203/rs.2.21231/v3 |
| Popis: | BackgroundTheEscherichia coliER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and had been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain was incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of theE. coliER2566 strain, and used high-throughput sequencing to explore how the strain adapted under external pressure.ResultsThe reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 190 hypothetical genes or pseudogenes, and resulted in the addition of 237 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 123 (63%) as coding genes. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3′ non-coding region) positioned 19-bp away from thelacI gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing.ConclusionThe ER2566 strain was used by both the general scientific community and the biotechnology industry. Reannotation of theE. coliER2566 strain not only improved the RefSeq data but uncovered a key site that might be involved in the transcription and translation of genes encoding the lactose operon repressor. We proposed that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study might facilitate a better understanding of gene function for the ER2566 strain under external burden and provided more clues to engineer bacteria for biotechnological applications. |
| Databáze: | OpenAIRE |
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