An efficient cellobiose-metabolizing strain of Escherichia coil would provide several industrial benefits, particularly with lignocellulosic biomass substrates. An efficient cellobiose-metabolizing E. coil strain (without the introduction of heterologous genes) was constructed by a combination of promoter engineering of cryptic operons (chb and asc) and adaptive evolution. Gene expression patterns in rapid and slow cellobiose-metabolizing strains were analyzed using DNA microarray. Genes involved in nucleotide biosynthesis, ATP generation, and flagellar assembly were upregulated following adaptation, indicating the possibility of active metabolism and fast growth. On the other hand, genes involved in chemotaxis toward glucose and response to starvation and other stresses were downregulated. No significant changes were observed in the expression of genes regulated by cyclic AMP receptor protein (CRP), the global regulator of carbon metabolism. Overexpression of ascB, which is located in the minor cryptic operon, seems to play a vital role in enhancing cellobiose metabolism. Changes in ascB transcription were confirmed by semiquantitative RT-PCR. The importance of ascB for cell growth on cellobiose was also validated by overexpression and knockout studies. Our study provides new insights into the role of the chb and asc operons in cellobiose metabolism. (C) 2012 Published by Elsevier Ltd.