title: fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity.
abstract: A major problem of long-chain fatty acid (LCFA) hydroxylation using Escherichia coli is that FadD (long-chain fatty acyl-CoA synthetase), which is necessary for exogenous LCFA transport, also initiates cellular consumption of LCFA. In this study, an effective method to prevent the cellular consumption of LCFA without impairing its transport is proposed. The main idea is that a heterologous enzyme which consumes LCFA can replace FadD in LCFA transport. For the model heterologous enzyme, CYP153A from Marinobacter aquaeolei, which converts palmitic acid into ω-hydroxy palmitic acid, was expressed in E. coli. When fadD was deleted from an E. coli strain, CYP153A indeed maintained the ability to transport LCFA. A disadvantage of fadD deletion mutant is the fact that FadD deficiency downregulates the transcription of fadL (outer membrane LCFA transporter) via FadR (fatty acid metabolism regulator protein), was solved by fadL overexpression from a plasmid. In addition, the overexpression of fadL was able to offset catabolite repression on fadL, allowing glucose to be used as the primary carbon source. In conclusion, the strain with fadD deletion and fadL overexpression showed 5.5-fold increase in productivity compared to the wild-type strain, converting 2.6 g/L (10.0 mM) of palmitic acid into 2.4 g/L (8.8 mM) of ω-hydroxy palmitic acid in a shake flask. This simple genetic manipulation can be applied to any LCFA hydroxylation using E. coli.
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea.
authors.forename: Jin H
authors.initials: JH
authors.lastname: Bae
authors.forename: Beom Gi
authors.initials: BG
authors.lastname: Park
authors.forename: Eunok
authors.initials: E
authors.lastname: Jung
authors.forename: Pyung-Gang
authors.initials: PG
authors.lastname: Lee
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D050612:Fatty Acid Transport Proteins / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D053520:Marinobacter / Q000201:enzymology / Q000235:genetics; D060847:Metabolic Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D050612:Fatty Acid Transport Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; C065691:fadL protein, E coli; D003066:Coenzyme A Ligases; C085934:acyl-coenzyme A synthetase, E coli