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Many bacteria possess all the machineries required to grow on fatty acids (FA) as a unique source of carbon and energy. FA degradation proceeds through the β-oxidation cycle that produces acetyl-CoA and reduced NADH and FADH cofactors. In addition to all the enzymes required for β-oxidation, FA degradation also depends on sophisticated systems for its genetic regulation and for FA transport. The fact that these machineries are conserved in bacteria suggests a crucial role in environmental conditions, especially for enterobacteria. Bacteria also possess specific enzymes required for the degradation of FAs from their environment, again showing the importance of this metabolism for bacterial adaptation. In this review, we mainly describe FA degradation in the Escherichia coli model, and along the way, we highlight and discuss important aspects of this metabolism that are still unclear. We do not detail exhaustively the diversity of the machineries found in other bacteria, but we mention them if they bring additional information or enlightenment on specific aspects.

Previously, we have developed an l -threonine-producing Escherichia coli strain TWF006 in which the regulator-encoding gene iclR was deleted. In this study, further modifications were performed on TWF006 to increase l -threonine yield. Firstly, the regulator-encoding gene fadR was deleted in TWF006, and the resulting strain TWF031 produced 18.86 g l -threonine from 30 g glucose after 24-h cultivation. Secondly, the regulator-encoding genes fabR and lacI in TWF031 were deleted, and the resulting strain TWF033 produced 19.21 g l -threonine from 30 g glucose after 24-h cultivation. Thirdly, additional copies of aceBA and fadBA were inserted into the lacZ locus of TWF033 and the native promoter of acs was replaced by the P tac-trc ; the resulting strain TWF038 produced 20.3 g l -threonine from 30 g glucose after 24-h cultivation. Finally, the genes ppnK , thrA*BC-rhtC , aspC , and ppc were inserted into the chromosome of TWF038; the resulting strain TWF044 produced 21.64 g l -threonine from 30 g glucose, or 28.49 g l -threonine from 40 g glucose after 24-h cultivation. After 48-h fed-batch fermentation, TWF044 produced 103.89 g/l l -threonine. The results suggest that coupling the fatty acid degradation and l -threonine biosynthesis pathway via the glyoxylate shunt could efficiently increase l -threonine production in E. coli .

The phagolysosomes of macrophages play a crucial role in eradicating pathogenic microorganisms, but bacteria have evolved sophisticated mechanisms to survive in the acidic environment of phagolysosomes, leading to host infection and subsequent dissemination. However, it is largely unknown how bacteria sense the extracellular stimuli and regulate their acid tolerance capacity to resist the killing by host immune cells. Here, we report the new substrate FadR of the serine/threonine kinase (STK) in Streptococcus suis serotype 2 (SS2) and demonstrate that the phosphorylation site is Thr230. Notably, FadR phosphorylation significantly enhances the acid resistance of SS2, leading to an increase in the lethality of SS2 in mice, and a marked increase in bacterial load in the blood and various organs, and more severe pathological changes in various organs of the mice. Interestingly, this study further indicated that FadR protein can bind to the promoter of arginine deiminase ( adi ), and FadR phosphorylation enhances its binding ability to the adi promoter and increases adi transcription levels. The increase of ADI in SS2 promotes the metabolism of arginine and increases the ammonia content, thus enhancing the acid resistance and intracellular survival capacity of the bacteria in macrophages. Altogether, the research reveals an acid resistance regulatory mechanism that bacteria can utilize the STK-FadR signaling axis to sense changes in the external acidic environment, and then manipulate the ADI system to enhance bacterial resistance to acidic environment or host immunity.

Protein FadR is known as a fatty acid metabolism global regulator that sustains cell envelope integrity by changing the profile of fatty acid. Here, we present its unique participation in the defense against reactive oxygen species (ROS) in the bacterium. FadR contributes to defending extracellular ROS by maintaining the permeability of the cell membrane. It also facilitates the ROS detoxification process by increasing the expression of ROS neutralizers (KatB, KatG, and AhpCF). FadR also represses the leakage of ROS by alleviating the respiratory action conducted by terminal cytochrome cbb3-type heme-copper oxidases (ccoNOQP). These findings suggest that FadR plays a comprehensive role in modulating the bacterial oxidative stress response, instead of merely strengthening the cellular barrier against the environment. This study sheds light on the complex mechanisms of bacterial ROS defense and offers FadR as a novel target for ROS control research.

Polyunsaturated fatty acids (PUFAs) play important roles in host immunity. Manipulation of lipid content in host tissues through diet or pharmacological interventions is associated with altered severity of various inflammatory diseases. The mammalian gastrointestinal tract is a complex biochemical organ that generates a diverse milieu of host- and microbe-derived metabolites. In this environment, bacterial pathogens sense and respond to specific stimuli, which are integrated into the regulation of their virulence programs. Previously, we identified the transcription factor FadR, a long-chain fatty acid (LCFA) acyl coenzyme A (acyl-CoA) sensor, as a novel virulence regulator in the human foodborne pathogen enterohemorrhagic Escherichia coli (EHEC). Here, we demonstrate that exogenous LCFAs directly inhibit the locus of enterocyte effacement (LEE) pathogenicity island in EHEC through sensing by FadR. Moreover, in addition to LCFAs that are 18 carbons in length or shorter, we introduce host-derived arachidonic acid (C 20:4 ) as an additional LCFA that is recognized by the FadR system in EHEC. We show that arachidonic acid is processed by the acyl-CoA synthetase FadD, which permits binding to FadR and decreases FadR affinity for its target DNA sequences. This interaction enables the transcriptional regulation of FadR-responsive operons by arachidonic acid in EHEC, including the LEE. Finally, we show that arachidonic acid inhibits hallmarks of EHEC disease in a FadR-dependent manner, including EHEC attachment to epithelial cells and the formation of attaching and effacing lesions. Together, our findings delineate a molecular mechanism demonstrating how LCFAs can directly inhibit the virulence of an enteric bacterial pathogen. More broadly, our findings expand the repertoire of ligands sensed by the canonical LFCA sensing machinery in EHEC to include arachidonic acid, an important bioactive lipid that is ubiquitous within host environments. IMPORTANCE Polyunsaturated fatty acids (PUFAs) play important roles in host immunity. Manipulation of lipid content in host tissues through diet or pharmacological interventions is associated with altered severity of various inflammatory diseases. Our work introduces a defined host-pathogen interaction by which arachidonic acid, a host-derived and dietary PUFA, can impact the outcome of enteric infection with the human pathogen enterohemorrhagic Escherichia coli (EHEC). We show that long-chain fatty acids including arachidonic acid act as signaling molecules that directly suppress a key pathogenicity island in EHEC following recognition by the fatty acyl-CoA-responsive transcription factor FadR. Thus, in addition to its established effects on host immunity and its bactericidal activities against other pathogens, we demonstrate that arachidonic acid also acts as a signaling molecule that inhibits virulence in an enteric pathogen.

As a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.

The improvement of bacterial tolerance to organic solvents is a main prerequisite for the microbial production of biofuels which are toxic to cells. For targeted genetic engineering of Escherichia coli to increase organic solvent tolerances (OSTs), we selected and investigated a total of 12 genes that participate in relevant mechanisms to tolerance. In a spot assay of 12 knockout mutants with n-hexane and cyclohexane, the genes fadR and marR were finally selected as the two key genes for engineering. Fatty acid degradation regulon (FadR) regulates the biosynthesis and degradation of fatty acids coordinately, and the multiple antibiotic resistance repressor (MarR) is the repressor of the global regulator MarA for multidrug resistance. In the competitive growth assay, the ΔmarR mutant became dominant when the pooled culture of 11 knockout mutants was cultivated successively in the presence of organic solvent. The increased OSTs in the ΔmarR and ΔfadR mutants were confirmed by a growth experiment and a viability test. The even more highly enhanced OSTs in the ΔfadR ΔmarR double mutant were shown compared with the two single mutants. Cellular fatty acid analysis showed that the high ratio of saturated fatty acids to unsaturated fatty acids plays a crucial role in OSTs. Furthermore, the intracellular accumulation of OST strains was significantly decreased compared with the wild-type strain.

The structure of FadR (VcFadR) complexed with the ligand oleoyl-CoA suggests an additional ligand-binding site. However, the fatty acid metabolism and its regulation is poorly addressed in , a species closely-related to . Here, we show crystal structures of FadR (ValFadR) alone and its complex with the palmitoyl-CoA, a long-chain fatty acyl ligand different from the oleoyl-CoA occupied by VcFadR. Structural comparison indicates that both VcFadR and ValFadR consistently have an additional ligand-binding site (called site 2), which leads to more dramatic conformational-change of DNA-binding domain than that of the FadR (EcFadR). Isothermal titration calorimetry (ITC) analyses defines that the ligand-binding pattern of ValFadR (2:1) is distinct from that of EcFadR (1:1). Together with surface plasmon resonance (SPR), electrophoresis mobility shift assay (EMSA) demonstrates that ValFadR binds , an important gene of unsaturated fatty acid (UFA) synthesis. The removal of from attenuates transcription and results in the unbalance of UFA/SFA incorporated into membrane phospholipids. Genetic complementation of the mutant version of (Δ42, 136-177) lacking site 2 cannot restore the defective phenotypes of Δ while the wild-type gene and addition of exogenous oleate can restore them. Mice experiments reveals that VcFadR and its site 2 have roles in bacterial colonizing. Together, the results might represent an additional example that illustrates the FadR-mediated lipid regulation and its role in pathogenesis.

Concerns about energy security and global petroleum supply have made the production of renewable biofuels an industrial imperative. The ideal biofuels are n-alkanes in that they are chemically and structurally identical to the fossil fuels and can “drop in” to the transportation infrastructure. In this work, an Escherichia coli strain that produces n-alkanes was constructed by heterologous expression of acyl-acyl carrier protein (ACP) reductase (AAR) and aldehyde deformylating oxygenase (ADO) from Synechococcus elongatus PCC7942. The accumulation of alkanes ranged from 3.1 to 24.0 mg/L using different expressing strategies. Deletion of yqhD, an inherent aldehyde reductase in E. coli, or overexpression of fadR, an activator for fatty acid biosynthesis, exhibited a nearly twofold increase in alkane titers, respectively. Combining yqhD deletion and fadR overexpression resulted in a production titer of 255.6 mg/L in E. coli, and heptadecene was the most abundant product.

The Escherichia coil fadR protein product, a paradigm/ prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid （UFA） biosynthesis. However the scenario in the other γ- proteobacteria, such as Shewanella with the marine ori- gin, is unusual in that Rodionov and coworkers predicted that only fabA （not fabB） has a binding site for FadR pro- tein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA- binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis （re- ferred to FadR_she） was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC （fast protein liquid chromatography） and chemical cross. linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobil. ity shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coil FadR （FadR_ec） in the ability of binding the E. coil fabA （and fabB） promoters. In an agreement with that of E. coil fabA, S. oneidensis fabA promoter bound both FadR_she andFadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyI-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native pro- moter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZtranscriptional fusion in E. coil. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by β-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid syn- thesis in the marine bacteria Shewanella genus.