Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

Abstract

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is thus of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl-CoA, we predicted amino acid positions within the effector binding pocket that would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.

A Rhodococcal Transcriptional Regulatory Mechanism Detects the Common Lactone Ring of AHL Quorum-Sensing Signals and Triggers the Quorum-Quenching Response

“…Besides, a putative binding site for FadR transcriptional factor was predicted upstream of the -35 box of qsdR promoter (Supplementary Figure S1 ). This sequence shares 75% identity with the consensus sequence recognized by FadR of E. coli ( Bacik et al, 2015 ).…”

“…The concomitant increasing in the intracellular amount of acyls-CoA molecules facilitates this phenomenon, since these molecules bind to the effector domain of the FadR regulator, causing a conformational change in the dimeric protein and the production of the QsdR repressor. This step is proposed according to in silico analysis of the genome of R. erythropolis strain R138 and the recent and well-documented bibliography related to the functioning of FadR regulator ( Bacik et al, 2015 ; My et al, 2015 ). (Black curved arrows and hairpin represents the initiation of gene transcription and a putative transcription terminator, respectively.)…”

“…In the E. coli model, FadR repression of fad genes expression is due to its binding to the promoter sites. This repression is lifted by interaction between FadR and long-chain acyls-CoA that have accumulated inside the cell ( Bacik et al, 2015 ; My et al, 2015 ). In Gram-positive bacteria, FadR also regulates several operons required for fatty acid degradation and recognizes fatty acyls-CoA as effectors ( Matsuoka et al, 2007 ).…”

CrgA Protein Represses AlkB2 Monooxygenase and Regulates the Degradation of Medium-to-Long-Chain n-Alkanes in Pseudomonas aeruginosa SJTD-1

“…It meant that the downstream intermediate could release the CrgA repressor and facilitate the transcription of AlkB2 monooxygenase. The release of this intermediate may due to the allosteric effect of CrgA caused by the effector binding (Bacik et al, 2015). Therefore, further work would be conducted to determine the structure of CrgA of P. aeruginosa SJTD-1 and the CrgA-DNA or CrgA-effector complex, and explore its recognition and interaction mechanism.…”

Degradation of Exogenous Fatty Acids in Escherichia coli

“…Mutagenesis experiments have permitted the dissection of the FadR ligand binding site in detail [ 5 , 89 ]. Using this information, it was then possible to obtain constitutive mutations that render the FadR variant insensitive to the addition of FA [ 89 , 90 ].…”