Fatty acid production and composition are determined by the type of acyl acyl carrier protein thioesterases (acyl-ACP TEs) expressed in Escherichia colt. Bacterial acyl-ACP TEs from Lactococcus lactis (SGJS47), Enterococcus faecalis (SGJS49), and Burkholderia cepacia (SGJS50) were codon-optimized and expressed in E. colt for enhanced fatty acid production. Samples were extracted at the lag, log, and stationary phases of cell growth, and gene expression levels of the codon optimized acy-ACP TEs as well as fatty acid production were monitored. At 24 h after initiation of gene expression, the ELITE expression level and fatty acid production in SGJS47 increased up to 15.8-fold and 3.2-fold compared to the control and other recombinant strains, respectively. Additionally, in SGJS47, improvement in free fatty acid (FFA) composition, high-specificity production of short-chain fatty acids (C8, C10) and unsaturated fatty acids (C16:1) was achieved in crude glycerol medium condition. Compared with control strain, the percentage of FFAs (C8 and C10) was enhanced by approximately 16- to 21-fold, C16:1 FFA ratio increased approximately 18-fold. Observation of codon-optimized acyl-ACP TE genes expression level in E. colt may be useful for understanding mechanisms towards improving fatty acid production. Engineered strains have the potential to overproduce specific FFAs and thereby reduce the cost of fatty acid production by using industrially inexpensive carbon sources. (C) 2014 Elsevier Inc. All rights reserved.