Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilispdc and adhB genes
Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilispdc and adhB genes
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大肠杆菌 KO11 基因组的光学图谱和测序揭示了广泛的染色体重排，以及运动发酵单胞菌 (Zymomonas mobilispdc) 和 adhB 基因的多个串联拷贝
DOI：
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发表时间：
2012
影响因子：
3.4
通讯作者：
L. Ingram
中科院分区：
文献类型：
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作者：
P. Turner;L. Yomano;L. Jarboe;L. Jarboe;S. W. York;Christy L. Baggett;Brélan E. Moritz;Emily B. Zentz;K. Shanmugam;L. Ingram
大肠杆菌 KO11 (ATCC 55124) 于 1990 年经过改造，通过将运动发酵单胞菌 (Zymomonas mobilispdc) 和 adhB 基因插入大肠杆菌 W (ATCC 9637) 的染色体来生产乙醇。 KO11FL（我们当前实验室版本的 KO11）及其亲本大肠杆菌 W 进行了测序，并使用光学 NcoI 限制性图谱作为模板将重叠群组装成基因组序列。大肠杆菌 W 含有质粒 pRK1 (102.5 kb) 和 pRK2 (5.4 kb)，但 KO11FL 仅含有 pRK2。用 AflII 和 BamHI 制作的 KO11FL 光学图显示串联重复区域，由至少 20 个 10 kb 单元的拷贝组成。重复区域位于 pdc、adhB 和氯霉素抗性基因的插入位点。这些基因的序列覆盖率比平均水平高约 25 倍，这与作为环化 DNA 插入的外源基因的扩增一致。选择较高水平的氯霉素抗性最初产生了具有较高 pdc 和 adhB 表达的菌株，从而通过增加基因拷贝数来改善发酵性能。 KO11 早期版本 ATCC 55124 的序列数据表明存在多个 pdc adhB 副本。 W 和 KO11FL 基因组的比较显示，KO11FL 中存在大量倒位和缺失，这主要是由 IS10 造成的，IS10 在 W 中不存在，但在 KO11FL 中存在于 30 个位点。早期KO11菌株ATCC 55124没有重排，仅包含一个IS10，并且缺乏KO11FL中存在的大多数积累的单核苷酸多态性（SNP）。尽管 KO11FL 中存在重排和 SNP，但发酵性能与 ATCC 55124 相同。
Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilispdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5 kb) and pRK2 (5.4 kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.