Regulation of cardiolipin biosynthesis by fatty acid transport protein-1 IN HEK 293 cells

Cardiolipin (CL) is a major phospholipid involved in energy metabolism mammalian mitochondria and fatty acid transport protein-1 (FATP-1) is a fatty acid transport protein that may regulate the intracellular level of fatty acyl-Coenzyme A's. Since fatty acids are required for oxidative phosphorylation via mitochondrial oxidation, we examined the effect of altering FATP-1 levels on CL biosynthesis. HEK-293 mock- and FATP-1 siRNA transfected cells or mock and FATP-1 expressing cells were incubated for 24 h with 0.1 mM oleic acid bound to albumin (1:1 molar ratio) then incubated for 24 h with 0.1 mM [1,3-³H]glycerol and radioactivity incorporated into CL determined. FATP-1 siRNA transfected cells exhibited reduced FATP-1 mRNA and increased incorporation of [1,3-³H]glycerol into CL (2-fold, p < 0.05) compared to controls indicating elevation in de novo CL biosynthesis. The reason for this was an increase in [1,3-³H]glycerol uptake and increase in activity and mRNA expression of the CL biosynthetic enzymes. In contrast, expression of FATP-1 resulted a reduction in incorporation of [1,3-³H]glycerol into CL (65%, p < 0.05) indicating reduced CL synthesis. [1,3-³H]Glycerol uptake was unaltered whereas activity of cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol synthetase (CDS) and CDS-2 mRNA expression were reduced in FATP-1 expressing cells compared to control. In addition, in vitro CDS activity was reduced by exogenous addition of oleoyl-Coenzyme A. The data indicate that CL de novo biosynthesis may be regulated by FATP-1 through CDS-2 expression in HEK 293 cells.     

Regulation of fatty acid metabolism by FadR is essential for Vibrio vulnificus to cause infection of mice

The opportunistic bacterial pathogen Vibrio vulnificus causes severe wound infection and fatal septicemia. We used alkaline phosphatase insertion mutagenesis in a clinical isolate of V. vulnificus to find genes necessary for virulence, and we identified fadR, which encodes a regulator of fatty acid metabolism. The fadR::mini-Tn5Km2phoA mutant was highly attenuated in a subcutaneously inoculated iron dextran-treated mouse model of V. vulnificus disease, was hypersensitive to the fatty acid synthase inhibitor cerulenin, showed aberrant expression of fatty acid biosynthetic (fab) genes and fatty acid oxidative (fad) genes, produced smaller colonies on agar media, and grew slower in rich broth than did the wild-type parent. Deletion of fadR essentially recapitulated the phenotypes of the insertion mutant, and the DeltafadR mutation was complemented in trans with the wild-type gene. Further characterization of the DeltafadR mutant showed that it was not generally hypersensitive to envelope stresses but had decreased motility and showed an altered membrane lipid profile compared to that of the wild type. Supplementation of broth with the unsaturated fatty acid oleate restored wild-type growth in vitro, and infection with oleate in the inoculum increased the ability of the DeltafadR mutant to infect mice. We conclude that fadR and regulation of fatty acid metabolism are essential for V. vulnificus to be able to cause disease in mammalian hosts.     

Mapping candidate genes for oleate biosynthesis and their association with unsaturated fatty acid seed content in soybean

The development of high-oleate soybean germplasm is hindered by the lack of knowledge of the genetic factors controlling oleate phenotypic variation. In the present study, several candidate genes implicated in oleate biosynthesis were mapped and their cosegregation with oleate, linoleate and linolenate quantitative trait loci (QTLs) was investigated. FAD2-2C, a previously described ω-6 desaturase isoform, was localized on linkage group E; whereas, a novel FAD2-2 isoform, designated as FAD2-2D, mapped on linkage group N. In addition, two isoforms were identified for the aminoalcoholphosphotransferase-encoding GmAAPT1 gene, denoted AAPT1a and AAPT1b. A database query suggested that only one functional copy of the FAD6 gene, encoding a plastid localized ω-6 desaturase, exists in the soybean genome. AAPT1a and FAD6 mapped on linkage group D1b, 23.40 cM apart. Linolenate QTLs with minor effects were identified near the FAD6 and AAPT1a markers in two segregating populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Role for fadR in unsaturated fatty acid biosynthesis in Escherichia coli

Escherichia coli K-12 mutants constitutive for the synthesis of the enzymes of fatty acid degradation (fad) synthesize significantly less unsaturated fatty acid (UFA) than do wild-type (fadR+) strains. The constitutive fadR mutants synthesize less UFA than do fadR+) strains both in vivo and in vitro. The inability of fadR strains to synthesize UFAs at rates comparable to those of fadR+ strains is phenotypically asymptomatic unless the fadR strain also carries a lesion in fabA, the structural gene for beta-hydroxydecanoyl-thioester dehydrase. Unlike fadR+ fabA(Ts) mutants, fadR fabA(Ts) strains synthesize insufficient UFA to support their growth even at low temperatures and, therefore, must be supplemented with UFA at both low and high temperatures. The low levels of UFA in fadR strains are not due to the constitutive level of fatty acid-degrading enzymes in these strains. These results suggest that a functional fadR gene is required for the maximal expression of UFA biosynthesis in E. coli.     

Regulation of fatty acid transport

PURPOSE OF REVIEW: The aim of the present review is to summarize recent developments in the area of regulation of fatty acid transport. RECENT FINDINGS: While controversy still exists regarding the contribution of passive diffusion versus protein-mediated fatty acid transport, both processes are now widely accepted. With the recent identification of an increasing number of putative fatty acid transporters, emphasis has been placed on regulation including fatty acid transport function of the protein, and also possible associated functions (acylCoA synthase activity and vectorial channelling to intracellular processing). Deciphering these issues has been facilitated through the use of loss-of-function (such as knockout) and gain-of-function (cell transfectants and transgenic mice) models. SUMMARY: It is likely that our concept of fatty acid transport will continue to converge, incorporating the individual functions of the wide variety of fatty acid transporters into an integrated physiologic framework with relevance to a number of diseases.     

Regulation of cyclopropane fatty acid biosynthesis by variations in enzyme activities

It is proposed that cyclopropane fatty acid biosynthesis in $\text{Lactobacillus plantarum}$ is regulated by $\text{in vivo}$ variations in the activities of two enzymes acting sequentially. S-adenosylhomocysteine hydrolase relieves the end-product inhibition of cyclopropane synthetase by degrading a product (S-adenosyl-homocysteine) of the latter enzyme activity. Both enzymes show an abrupt increase and subsequent decrease in activity at a time during the bacterial growth cycle which corresponds to the period of most rapid synthesis of cyclopropane fatty acid $\text{in vivo}$.     

Regulation of fatty acid biosynthesis in Escherichia coli.

Our understanding of fatty acid biosynthesis in Escherichia coli has increased greatly in recent years. Since the discovery that the intermediates of fatty acid biosynthesis are bound to the heat-stable protein cofactor termed acyl carrier protein, the fatty acid synthesis pathway of E. coli has been studied in some detail. Interestingly, many advances in the field have aided in the discovery of analogous systems in other organisms. In fact, E. coli has provided a paradigm of predictive value for the synthesis of fatty acids in bacteria and plants and the synthesis of bacterial polyketide antibiotics. In this review, we concentrate on four major areas of research. First, the reactions in fatty acid biosynthesis and the proteins catalyzing these reactions are discussed in detail. The genes encoding many of these proteins have been cloned, and characterization of these genes has led to a better understanding of the pathway. Second, the function and role of the two essential cofactors in fatty acid synthesis, coenzyme A and acyl carrier protein, are addressed. Finally, the steps governing the spectrum of products produced in synthesis and alternative destinations, other than membrane phospholipids, for fatty acids in E. coli are described. Throughout the review, the contribution of each portion of the pathway to the global regulation of synthesis is examined. In no other organism is the bulk of knowledge regarding fatty acid metabolism so great; however, questions still remain to be answered. Pursuing such questions should reveal additional regulatory mechanisms of fatty acid synthesis and, hopefully, the role of fatty acid synthesis and other cellular processes in the global control of cellular growth.     

Regulation of fatty acid biosynthesis by hydrocarbon substrates in Mycobacterium convolutum.

When Mycobacterium convolutum R22 was grown on the n-alkanes C13 through C16, the predominant fatty acids were of the same chain length as the growth substrate. Cells grown on C13 through C16 n-alkanes incorporated between 15 and 85 pmol of acetate per microgram of lipid into the fatty acids, whereas acetate- or propane-grown cells incorporated 280 and 255 pmol of acetate per microgram of lipid, respectively. In vivo experiments demonstrated that hexadecane, hexadecanoic acid, and hexadecanoylcoenzyme A (CoA) all inhibited de novo fatty acid synthesis. Hexadecanoyl-CoA was the most potent inhibitor. Hexadecane and hexadecanoic acid inhibited acetyl-CoA carboxylase by up to 37 and 39%, respectively, at 1 mM. Hexadecanoyl-CoA inhibited the enzyme activity by 65% at 50 micrometer. Cells that were grown on C14 through C16 n-alkanes had about 25 times less acetyl-CoA carboxylase activity than did cells grown on acetate or propane, suggesting repressed levels of the enzyme. Hexadecane- or pentadecane-grown cells were found to have 5 to 10 times more intracellular free fatty acid than cells grown on acetate, propane, or ethane.     

Increasing unsaturated fatty acid contents in Escherichia coli by coexpression of three different genes

Biodiesel is an interesting alternative energy source and is used as substitute for petroleum-based diesel. Microorganisms have been used for biodiesel production due to their significant environmental and economic benefits. However, few researches have investigated the regulation of fatty acid composition of these microbial diesels. Fatty acid biosynthesis in Escherichia coli has provided a paradigm for other bacteria and plants. By overexpressing two genes (fabA and fabB) associated with unsaturated fatty acid (UFA) synthesis in E. coli, we have engineered an efficient producer of UFAs. Saturated fatty acid (SFA) contents decreased from 50.2% (the control strain) to 34.6% (the recombinant strain overexpressing fabA and fabB simultaneously) and the ratio of cis-vaccenate (18:1Δ11), a major UFA in E. coli, reached 51.1% in this recombinant strain. When an Arabidopsis thaliana thioesterase (AtFatA) was coexpressed with these two genes, 0.19 mmol l⁻¹ fatty acids was produced by this E. coli strain after 18-h culture under shake-flask conditions. Free fatty acids made up about 37.5% of total fatty acid concentration in this final engineered strain carrying fabA, fabB, and AtFatA, and the ratio of UFA/SFA reached 2.3:1. This approach offers a means to improve the fatty acid composition of microdiesel and might pave the way for production of biodiesel equivalents using engineered microorganisms in the near future.