This study devised a pathway in Escherichia coli for direct production of long chain hydroxyfatty acids (HFAs) from glucose. This is first report on the biosynthesis of HFAs from renewable sugar, without the need of exogenous fatty acids. By employing thioesterases BTE and 'TesA to tailor the composition of free fatty acids (FFAs) and using fatty acid hydroxylase P450(BM3) to convert FFAs to HFAs, high-specificity production of C12 and C14 HFAs was achieved. By further knocking out the endogenous fadD gene of E. coli, an engineered strain capable of producing 117.0 mg/L HFAs was finally obtained, representing a high HFA production in shake flask. This study indicated an attractive metabolic strategy for the biosynthesis of HFAs directly from renewable carbohydrates resources. (C) 2012 Elsevier Ltd. All rights reserved.