Improvement of butanol production by Escherichia coli using Tn5 transposon

For engineering an efficient butanol-producing E. coli strain, many efforts have been paid on the known genes or pathways based on current knowledge. It is reasonable to believe that many genes in the genome could also contribute to butanol production in an unexpected way. In this work, we used the Tn5 transposon to construct a mutant library including 1 196 strains in a previously engineered butanol-producing E. coli strain. To screen the strains with improved titer of butanol production, we developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader. The principle is that pyruvate is the precursor of butanol and its concentration was found to be inversely correlative with butanol in the fermentation broth. Using this method, we successfully screened three mutants with increased butanol titer. The insertion sites of Tn5 transposon was in the ORFs of pykA, tdk, and cadC by inverse PCR and sequencing. These found genes would be efficient targets for further strain improvement. And the genome scanning strategy described here will be helpful for other microbial cell factory construction.