Enhanced free fatty acid production by codon-optimized Lactococcus lactis acyl-ACP thioesterase gene expression in Escherichia coli using crude glycerol

• To reduce cost of biodiesel, we used crude glycerol as a carbon source for production of fatty acid.
• Fatty acid production of E. coli SGJS47 (overexpressing the OPLlTE gene) increased up to 3.2-fold.
• The percentage of FFAs was enhanced by 16- to 21-fold, C16:1 FFA increased 18-fold in recombinants.
• OPLlTE is a useful enzyme for optimizing the production and composition of FFAs.

Fatty acid production and composition are determined by the type of acyl–acyl carrier protein thioesterases (acyl-ACP TEs) expressed in Escherichia coli. Bacterial acyl-ACP TEs from Lactococcus lactis (SGJS47), Enterococcus faecalis (SGJS49), and Burkholderia cepacia (SGJS50) were codon-optimized and expressed in E. coli 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 OPLlTE 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. coli 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.