Next-generation sequencing technologies are revolutionizing the pharmaceutical development. These technologies can not only be used in the drug discovery process but also for strain improvement in the manufacturing process. Using short read sequencing technology, we sequenced a parent strain and a hypersecreter strain (B41) of W3110 E. coli. B41 strain was created by chemically mutagenising the parent strain and secretes four-fold more active hemolysin protein (HlyA) relative to the parent strain via Type-I pathway. An average of 26.9x coverage of the two genomes resulted in a single G to T polymorphism in B41 strain at position 1,074,787 of the genome. The missense mutation results in translation termination of a transcriptional regulator RutR, coded by ycdc gene. The mRNA expression profiling data for the two strains also suggest significant expression change of the genes regulated by RutR, which is consistent with the observed mutation. We also tested the secretion phenotype in ycdc deletion mutant and observed a similar increase in active HlyA secretion relative to the parent strain.