Tuesday, May 3, 2011
E. coli KO11 was engineered to produce ethanol by inserting the pyruvate decarboxlase (pdc) and alcohol dehydrogenase (adhB) genes from Zymomonas mobilis. Production of succinate, an unwanted byproduct, was eliminated by mutating fumarate reductase (frd). The KO11 genome was sequenced by 454 technology and assembled with Newbler to produce 196 contigs. An optical NcoI restriction map of the entire KO11 genome was generated to enable closure of the contigs. Gaps were filled by sequencing PCR products extending between adjoining contig ends. The optical map of KO11 showed an apparent tandem repeat region, which was confirmed by additional AflII and BamHI maps of KO11. Each repeat was ~10 kb in length, and present in approximately 20 copies. The repeat region was in between contigs that flanked the pflB gene, which was used as the insertion site for the pdc, adhB and chloramphenicol resistance (cat) genes. The sequence coverage of contigs lying within the pdc, adhB and cat genes was about 20-fold higher than the average sequence coverage, consistent with tandem duplication of the foreign genes, which were inserted as a circularized DNA fragment. Selection for higher levels of chloramphenicol-resistance originally allowed isolation of strains with higher expression of both pdc and adhB, and hence improved fermentation performance, by increasing the number of gene copies. The chromosomal sequence of the parent strain ATCC 9637 (W) has also been completely assembled, showing extensive rearrangements in KO11, and enabling detection of changes in KO11 that may contribute to improved fermentation properties.