Pseudomonas aeruginosa Directly Shunts β-Oxidation Degradation Intermediates into De Novo Fatty Acid Biosynthesis

We identified the fatty acid synthesis (FAS) initiation enzyme in Pseudomonas aeruginosa as FabY, a β-ketoacyl synthase KASI/II domain-containing enzyme that condenses acetyl coenzyme A (acetyl-CoA) with malonyl-acyl carrier protein (ACP) to make the FAS primer β-acetoacetyl-ACP in the accompanying article (Y. Yuan, M. Sachdeva, J. A. Leeds, and T. C. Meredith, J. Bacteriol. 194:5171-5184, 2012). Herein, we show that growth defects stemming from deletion of fabY can be suppressed by supplementation of the growth media with exogenous decanoate fatty acid, suggesting a compensatory mechanism. Fatty acids eight carbons or longer rescue growth by generating acyl coenzyme A (acyl-CoA) thioester β-oxidation degradation intermediates that are shunted into FAS downstream of FabY. Using a set of perdeuterated fatty acid feeding experiments, we show that the open reading frame PA3286 in P. aeruginosa PAO1 intercepts C(8)-CoA by condensation with malonyl-ACP to make the FAS intermediate β-keto decanoyl-ACP. This key intermediate can then be extended to supply all of the cellular fatty acid needs, including both unsaturated and saturated fatty acids, along with the 3-hydroxyl fatty acid acyl groups of lipopolysaccharide. Heterologous PA3286 expression in Escherichia coli likewise established the fatty acid shunt, and characterization of recombinant β-keto acyl synthase enzyme activity confirmed in vitro substrate specificity for medium-chain-length acyl CoA thioester acceptors. The potential for the PA3286 shunt in P. aeruginosa to curtail the efficacy of inhibitors targeting FabY, an enzyme required for FAS initiation in the absence of exogenous fatty acids, is discussed.     

Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene

Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC₅₀) of the enzyme activity was measured to be 400 M, whereas the IC₅₀ value was 15 M for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 pheno-type and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly Ser) in the subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.     

Cloning and sequence analysis of putative type II fatty acid synthase genes from <Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.

The cultivated peanut is a valuable source of dietary oil and ranks fifth among the world oil crops. Plant fatty acid biosynthesis is catalysed by type II fatty acid synthase (FAS) in plastids and mitochondria. By constructing a full-length cDNA library derived from immature peanut seeds and homology-based cloning, candidate genes of acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, β-ketoacyl-ACP synthase (I, II, III), β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were isolated. Sequence alignments revealed that primary structures of type II FAS enzymes were highly conserved in higher plants and the catalytic residues were strictly conserved in Escherichia coli and higher plants. Homologue numbers of each type II FAS gene expressing in developing peanut seeds varied from 1 in KASII, KASIII and HD to 5 in ENR. The number of single-nucleotide polymorphisms (SNPs) was quite different in each gene. Peanut type II FAS genes were predicted to target plastids except ACP2 and ACP3. The results suggested that peanut may contain two type II FAS systems in plastids and mitochondria. The type II FAS enzymes in higher plants may have similar functions as those in E. coli.     

Resistance to azolic compounds in clinical Trichophyton spp. strains

The increase of dermathophytosis in patients with poor therapeutic response leads us to study the antifungal susceptibility of 36 clinical isolates to itraconazole, ketoconazole and fluconazole by the E-test method. According to established parameters by the Clinical Laboratory Standards Institute, the resistance to one or more antifungal drugs was demonstrated in seven isolates (19.4%) as follows: three Trichophyton rubrum, three T. mentagrophytes and one T. tonsurans. A T. rubrum isolate was resistant to the three azolic drugs; the other six only to fluconazole. It is important to establish the antifungal susceptibility as part of the study procedures in patients with dermatophytosis and a poor antifungal response.     

Coordinate genetic control of yeast fatty acid synthase genes FAS1 and FAS2 by an upstream activation site common to genes involved in membrane lipid biosynthesis.

A systematic search for upstream controlling elements necessary for efficient expression of the yeast fatty acid synthase genes FAS1 and FAS2 revealed identical activation sites, UASFAS, in front of both FAS genes. The individual element confers, in a heterologous yeast test system, an approximately 40-fold stimulation of basal gene expression. The UASFAS motifs identified have the consensus sequence TYTTCACATGY and function in either orientation. The same sequence motif is found in the upstream regions of all so far characterized yeast genes encoding enzymes of phospholipid biosynthesis. In gel retardation assays, a protein factor, Fbf1 (FAS binding factor), was identified which interacted with UASFAS. The UASFAS motif proved to be an inositol/choline responsive element (ICRE) conferring strict repression by exogenous inositol and choline on a heterologous reporter gene. Its core sequence perfectly matches the CANNTG motif typical of basic helix-loop-helix DNA-binding proteins. In contrast to the individual UASFAS element, the intact yeast FAS promoters are not significantly influenced by inositol and choline, and thus allow nearly constitutive fatty acid synthase production. Available evidence suggests that additional cis- and trans-acting elements, other than UASFAS and Fbf1, are involved in this constitutive FAS gene expression.     

Starter unit specificity directs genome mining of polyketide synthase pathways in fungi

Search of the protein database with the aflatoxin pathway polyketide synthase (PKS) revealed putative PKSs in the pathogenic fungi Coccidioides immitis and Coccidioides posadasii that could require partnerships with a pair of fatty acid synthase (FAS) subunits for the biosynthesis of fatty acid-polyketide hybrid metabolites. A starter unit:acyl-carrier protein transacylase (SAT) domain was discovered in the nonreducing PKS. This domain is thought to accept the fatty acid product from the FAS to initiate polyketide synthesis. We expressed the C. immitis SAT domain in Escherichia coli and showed that this domain, unlike that from the aflatoxin pathway PKS, transferred octanoyl-CoA four times faster than hexanoyl-CoA. The SAT domain also formed a covalent octanoyl intermediate and transferred this group to a free-standing ACP domain. Our results suggest that C. immitis/posadasii, both human fungal pathogens, contain a FAS/PKS cluster with functional similarity to the aflatoxin cluster found in Aspergillus species. Dissection of the PKS and determination of in vitro SAT domain specificity provides a tool to uncover the growing number of similar sequenced pathways in fungi, and to guide elucidation of the fatty acid-polyketide hybrid metabolites that they produce.