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.

Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo

“…Recently, bacterial CYPs in the CYP153A family, such as CYP153A16 from Mycobacterium marinum, CYP153A33 from Marinobacter aquaeolei, and CYP153A34 from Polaromonas sp., were characterized as fatty acid ω-hydroxylases, which act on saturated and unsaturated fatty acids (Honda Malca et al 2012). Using CYP153A33 in recombinant E. coli system, high ω-regioselective bioconversion of 1.2 g/L 12-hydroxy dodecanoic acid from 10.0 g/L (50 mM) dodecanoic acid and 4.0 g/L 12-hydroxy dodecanoic acid methyl ester from 174 g/L dodecanoic acid methyl ester was achieved by overexpression of AlkL, the fatty acid ester transport (Scheps et al 2013), and bioconversion of 2.4 g/L 16-hydroxy palmitic acid from 2.6 g/L (10 mM) palmitic acid was also reported by deletion of a fatty acid ω-oxidation degradation pathway (i.e., ΔfadD) and overexpression of a fatty acid transporter, FadL (Bae et al 2014). Despite such successful ω-HFA production in E. coli, there are still rooms for enhancing both yield and productivity of long chain (C12-C18) ω-HFAs by screening higher catalytically active CYPs among numerous CYP sequences and incorporating an appropriate electron transfer system into CYPs.…”

“…pCamB-CYP153A35, pCamB-CamA, pCamB-CYP153A35-CamA, and pCamB-CamA-CYP153A35 encoding cyp153A35 and CamAB as an operon were constructed by compatible cohesive ends produced by SpeI and XbaI. We previously cloned CYP153A33 from M. aquaeolei and fadL from E. coli into pCDFmT7 (Bae et al 2014), and CamA and CamB from Pseudomonas putida were cloned into pET28a and pETDuet-1, respectively (Choi et al 2010).…”

P212A Mutant of Dihydrodaidzein Reductase Enhances ( S )-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

“…Enzymatically synthesized THD enantiomers were analyzed and confirmed via HPLC with a chiral column. The THD enantiomers were then derivatized with N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) at 50°C for 5 min and were analyzed with a gas chromatograph-mass spectrometer (GC-MS) equipped with an electron impact (EI) ionization source for identification according to a previously described method (25), with some modifications. The GC oven temperature started at 65°C, was held for 5 min, and then increased by 3°C/min to 250°C, with holding for 10 min.…”

Boosting the hydroxyfatty acid synthesis inEscherichia coliby expression ofBacillus megateriumglucose dehydrogenase

“…The poor HFA production of WX5 may be attributed to low adenosine triphosphate (ATP), or membrane lipid during hydroxylation in E. coli. [12] The chemical compositions of HFAs produced by WX5 did not make much change. The results are not surprising, because the deletion of fadD only enhanced the quantity, but not the composition of FFAs.…”

“…However, this method needs a long reaction time (36 h) and the fed-batch reaction in the process limits its further industrial application. [12] Wang et al [13] reported an engineered E. coli could produce HFAs directly from glucose, but the production was low (117.0 mg/L). All these drawbacks led people to search for more effective biosynthesis pathway that may contribute to a further improvement of HFAs production.…”