Fatty Acid Degradation in Escherichia coli

Abstract

1. An enzymatic activity is described in oleate-grown cells of E. coli, which has the cofactor requirements of a long chain acid : CoA ligase (AMP). This enzyme is a t least partially membrane bound and activates saturated and mono-and polyunsaturated fatty acids. It is inactive with butyrate and hexanoate. The enzyme is induced by oleate under similar conditions, but a t different relative rates, as three other enzymes of fatty acid degradation (thiolase, crotonase, hydroxyacylCoA dehydrogenase) which are under coordinate control. 2.A mutant lacking acyl-CoA synthetase synthesizes the other three enzymes at the basal rate of the wild type in the presence or absence of oleate in the medium. This suggests that the fatty acyl-CoA derivatives rather than the fatty acids serve as inducers. The same mutant is unable to incorporate fatty acids into bacterial lipids. The acyl-CoA synthetase is therefore required for lipid synthesis from exogenous fatty acids in vivo.3. The isolation and properties of mutants unable to degrade oleate are described. A pleiotropic mutant lacking thiolase, crotonase and hydroxyacyl-CoA dehydrogenase still contains the inducible acyl-CoA synthetase. Mutants lacking thiolase and hydroxyacyl-CoA dehydrogenase have inducible wild type levels of the other enzymes.4. Mutants affecting the synthesis of thiolase, hydroxyacyl-CoA dehydrogenase or crotonase map between the loci metE and rha on the E . coli-chromosome and are probably closely linked. The mutant lacking acyl-CoA synthetase is not linked to the other mutants and maps near the his-locus.5. Only fatty acids with more than twelve carbon atoms serve as inducers or can be used as sole carbon source by the wild type. This specificity is therefore typical for the abolition of repression by the interaction with the fatty acids.6. Spontaneous mutants can be isolated which grow on decanoate. These are constitutive for the synthesis of all four enzymes investigated and can only grow on fatty acids with more than eight carbon atoms. Thus, after destruction of the repression mechanism the growth behavior is a reflection of the substrate specificity of the acyl-CoA synthetase.7. Starting with the constitutive mutants, spontaneous mutants have been isolated which can grow on butyrate. These fall into two classes: (a) One class grows on butyrate and poorly on long chain fatty acids. It makes all four enzymes constitutively but has twenty times more thiolase activity than the parent. This thiolase activity is not due to a gene linked to metE. (b) The other class can grow on butyrate and fatty acids with more than eight carbon atoms. It synthesizes the enzymes constitutively.8. The data suggest that the expression of the linked structural genes of crotonase, hydroxyacyl-CoA dehydrogenase and thiolase and the unlinked gene of the acyl-CoA synthetase form a regulon which is controlled by a common regulator gene.

The Escherichia coli fadK (ydiD) Gene Encodes an Anerobically Regulated Short Chain Acyl-CoA Synthetase

“…These results indicated that FadK could functionally replace FadD under anaerobic conditions. However, fadD mutants are unable to grow on long chain fatty acids aerobically (8,18,19) and thus the failure of FadK to replace FadD under aerobic conditions was attributed to insufficient expression of FadK or to impairment of enzyme function.…”

Unexpected Functional Diversity among FadR Fatty Acid Transcriptional Regulatory Proteins

“…Addition of long chain (ϾC 12 ) fatty acids induces the fad enzymes, whereas medium (C 7 -C 11 ) or short chain-length (C 4 -C 6 ) fatty acids cannot (11,12). Therefore, wild-type E. coli strains utilize long-chain fatty acids such as oleate (C18:1), but not medium-chain-length fatty acids such as decanoate (C10:0) as sole carbon and energy sources.…”

“…Therefore, wild-type E. coli strains utilize long-chain fatty acids such as oleate (C18:1), but not medium-chain-length fatty acids such as decanoate (C10:0) as sole carbon and energy sources. However, mutants that grow on decanoate are readily isolated by plating wild-type cells onto minimal media containing this acid as sole carbon source (11,12). These stains have constitutive levels of the fad enzymes and have mutations that map in fadR (2,11,13).…”

Nitrile Pathway Involving Acyl-CoA Synthetase

“…Next, we examined whether or not AcsA complements the FadD (longchain acyl CoA synthetase)-deficient strain (K27). In contrast Overall Nitrile Pathway Involving Acyl-CoA Synthetaseto the wild-type strain that grew in the medium containing a long-chain fatty acid (oleate) as the sole carbon and energy source, the K27 strain did not grow (38,43). We transformed the K27 strain with pUC-acsA and plated the transformants on glucose or oleate minimal agar plates, respectively.…”

“…E. coli BL21-CodonPlus(DE3)-RIL (Novagen, Madison, WI) was used as the host for one plasmid, pET-24a(ϩ) (Novagen), and its derivative, and it was also used for expression of the acyl-CoA synthetase gene (acsA). E. coli acetyl-CoA synthetase-deficient mutant strain AJW1781 (CGSC#8076; ⌬acs-1::kan thr-1 leuB6(Am) ⌬(codB-lacI)3 rpsL136 metF159(Am) thi-1) (37) and long-chain acyl-CoA synthetase-deficient mutant strain K27 (CGSC#5478; fadD88) (38) DNA Manipulations-Restriction endonucleases, DNA polymerase, and T4 DNA ligase were purchased from Toyobo Co., Ltd. (Osaka, Japan). Nucleotides were sequenced by the dideoxy-chain terminating method using an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA).…”