The transcriptome profiles of recombinant Klebsiella pneumoniae producing human insulin-like growth factor I fusion protein (IGF-I_f) during the high-cell-density fed-batch culture were analyzed using DNA microarrays. The expression levels of 643 genes were significantly altered after induction.It was found out that about 302 genes were significantly down-regulated during the production of IGF-I_f after induction. Among these down-regulated genes, we rationally selected and coexpressed in K. pneumoniae producing IGF-I_f the prsA gene (encoding a phosphoribosyl pyrophosphate synthetase) and the glpF gene (encoding a glycerol transporter), which are involved in an early key step in the biosynthetic pathway of nucleotides and amino acids (Trp and His) and the first step in glycerol utilization, respectively. As a result, the production of IGF-I_f could be increased from 3.7 ± 0.14 (± standard deviation) to 5.4 ± 0.o9 g/liter. The volumetric productivity was also increased from 0.36 ± 0.027 to 0.82 ± 0.048 g/liter/h. These results demonstrate that transcriptome profiling can provide invaluable information in designing engineered strains showing enhanced performance.