Hydroxylated fatty acids (HFAs) are used as important precursors for bulk and fine chemicals in the chemical industry. Here, to overproduce long-chain (C16-C18) fatty acids and hydroxy fatty acid, their biosynthetic pathways including thioesterase (Lreu_0335) from Lactobacillus reuteri DSM20016, beta-hydroxyacyl-ACP dehydratase (fabZ) from Escherichia coli, and a P450 system (i.e., CYP153A from Marinobacter aquaeolei VT8 and camA/camB from Pseudomonas putida ATCC17453) were overexpressed. Acyl-CoA synthase (fadD) involved in fatty acid degradation by beta-oxidation was also deleted in E. coli BW25113. The engineered E. coli FFA4 strain without the P450 system could produce 503.0 mg/l of palmitic (C-16) and 508.4 mg/l of stearic (C-18) acids, of which the amounts are ca. 1.6- and 2.3-fold higher than those of the wild type. On the other hand, the E. coli HFA4 strain including the P450 system for omega-hydroxylation could produce 211.7 mg/l of omega-hydroxy palmitic acid, which was 42.1 +/- 0.1 % of the generated palmitic acid, indicating that the hydroxylation reaction was the rate-determining step for the HFA production. For the maximum production of omega-hydroxy palmitic acid, NADH, i.e., an essential cofactor for P450 reaction, was overproduced by the integration of NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii into E. coli chromosome and the deletion of alcohol dehydrogenase (ADH). Finally, the NADH-level-optimized E. coli strain produced 610 mg/l of omega-hydroxy palmitic acid (omega-HPA), which was almost a threefold increase in its yield compared to the same strain without NADH overproduction.