Impact of various ß-ketothiolase genes on PHBHHx production in Cupriavidus necator H16 derivatives.

Poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] (PHBHHx) is a type of biopolyester of the polyhydroxyalkanoate group (PHA). Due to a wide range of properties resulting from the alteration of the (R)-3-hydroxyhexanoate (3HHx) composition, PHBHHx is getting a lot of attention as a substitute to conventional plastic materials for various applications. Cupriavidus necator H16 is the most promising PHA producer and has been genetically engineered to produce PHBHHx efficiently for many years. Nevertheless, the role of individual genes involved in PHBHHx biosynthesis is not well elaborated. C. necator H16 possesses six potential physiologically active ß-ketothiolase genes identified by transcriptome analysis, i.e., phaA, bktB, bktC (h16_A0170), h16_A0462, h16_A1528, and h16_B0759. In this study, we focused on the functionality of these genes in vivo in relation to 3HHx monomer supply. Gene deletion experiments identified BktB and H16_A1528 as important ß-ketothiolases for C6 metabolism in ß-oxidation. Furthermore, in the bktB/h16_A1528 double-deletion strain, the proportion of 3HHx composition of PHBHHx produced from sugar was very low, whereas that from plant oil was significantly higher. In fact, the proportion reached 36.2 mol% with overexpression of (R)-specifc enoyl-CoA hydratase (PhaJ) and PHA synthase. Furthermore, we demonstrated high-density production (196 g/L) of PHBHHx with high 3HHx (32.5 mol%) by fed-batch fermentation with palm kernel oil. The PHBHHx was amorphous according to the differential scanning calorimetry analysis. KEY POINTS: • Role of six ß-ketothiolases in PHBHHx biosynthesis was investigated in vivo. • Double-deletion of bktB/h16_A1528 results in high 3HHx composition with plant oil. • Amorphous PHBHHx with 32.5 mol% 3HHx was produced in high density by jar fermenter.

Increase in omega-6 and decrease in omega-3 polyunsaturated fatty acid oxidation elevates the risk of exudative AMD development in adults with Chinese diet.

Appropriate diet is essential for the regulation of age-related macular degeneration (AMD). In particular the type of dietary polyunsaturated fatty acids (PUFA) and poor antioxidant status including carotenoid levels concomitantly contribute to AMD risk. Build-up of oxidative stress in AMD induces PUFA oxidation, and a mix of lipid oxidation products (LOPs) are generated. However, LOPs are not comprehensively evaluated in AMD. LOPs are considered biomarkers of oxidative stress but also contributes to inflammatory response. In this cross-sectional case-control study, plasma omega-6/omega-3 PUFA ratios and antioxidant status (glutathione, superoxide dismutase and catalase), and plasma and urinary LOPs (41 types) were determined to evaluate its odds-ratio in the risk of developing exudative AMD (n = 99) compared to age-gender-matched healthy controls (n = 198) in adults with Chinese diet. The odds ratio of developing exudative AMD increased with LOPs from omega-6 PUFA and decreased from those of omega-3 PUFA. These observations were associated with a high plasma omega-6/omega-3 PUFA ratio and low carotenoid levels. In short, poor PUFA and antioxidant status increased the production of omega-6 PUFA LOPs such as dihomo-isoprostane and dihomo-isofuran, and lowered omega-3 PUFA LOPs such as neuroprostanes due to the high omega-6/omega-3 PUFA ratios; they were also correlated to the risk of AMD development. These findings indicate the generation of specific LOPs is associated with the development of exudative AMD.

Proteomic analysis reveals Xuesaitong injection attenuates myocardial ischemia/reperfusion injury by elevating pyruvate dehydrogenase-mediated aerobic metabolism.

Xuesaitong injection (XST), which mainly consists of Panax notoginseng saponins, has been widely used for treating cardio-cerebral vascular diseases. However, the underlying mechanisms of XST associated with its cardioprotective effects are still unclear. To identify the potential target proteins of XST, two-dimensional gel electrophoresis (2-DE)-based proteomics was utilized to analyze the protein profile of myocardium in rats with myocardial ischemia/reperfusion (I/R) injury. The differentially expressed proteins were identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometry. It is interesting that XST can alter the expression of 7 proteins, including pyruvate dehydrogenase E1 alpha (PDHA1), hydroxyacyl-coenzyme A dehydrogenase (HADHA), peroxiredoxin 3 (PRX3), gamma-enolase, acetyl-coenzyme A acyltransferase 2 (ACAA2), etc. Functional analysis revealed that those proteins were chiefly related to cardiac energy metabolism and oxidative stress. The cardioprotective effects of XST were further validated in H9c2 cardiac muscle cells with hypoxia/reoxygenation injury. We found that XST can promote the activity of PDH, an important enzyme related to the TCA cycle, as well as increase the intracellular content of acetyl-CoA and ATP. Moreover, XST also attenuated intracellular MDA release in H2O2-injured cardiac cells. This is the first study on the proteomic expression of XST-treated myocardium with I/R injury to reveal that the cardioprotective effects of XST may be attributed to the PDH-mediated restoration of aerobic glucose oxidation.

Polymorphisms in genes involved in fatty acid ß-oxidation interact with dietary fat intakes to modulate the plasma TG response to a fish oil supplementation.

A large inter-individual variability in the plasma triglyceride (TG) response to an omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation has been observed. The objective was to examine gene-diet interaction effects on the plasma TG response after a fish oil supplementation, between single-nucleotide polymorphisms (SNPs) within genes involved in fatty acid ß-oxidation and dietary fat intakes. Two hundred and eight (208) participants were recruited in the greater Quebec City area. The participants completed a six-week fish oil supplementation (5 g fish oil/day: 1.9-2.2 g EPA and 1.1 g DHA). Dietary fat intakes were measured using three-day food records. SNPs within RXRA, CPT1A, ACADVL, ACAA2, ABCD2, ACOX1 and ACAA1 genes were genotyped using TAQMAN methodology. Gene-diet interaction effects on the plasma TG response were observed for SNPs within RXRA (rs11185660, rs10881576 and rs12339187) and ACOX1 (rs17583163) genes. For rs11185660, fold changes in RXRA gene expression levels were different depending on SFA intakes for homozygotes T/T. Gene-diet interaction effects of SNPs within genes involved in fatty acid ß-oxidation and dietary fat intakes may be important in understanding the inter-individual variability in plasma TG levels and in the plasma TG response to a fish oil supplementation.

A plant thiolase involved in benzoic acid biosynthesis and volatile benzenoid production.

The exact biosynthetic pathways leading to benzoic acid (BA) formation in plants are not known, but labeling experiments indicate the contribution of both beta-oxidative and non-beta-oxidative pathways. In Petunia hybrida BA is a key precursor for the production of volatile benzenoids by its flowers. Using functional genomics, we identified a 3-ketoacyl-CoA thiolase, PhKAT1, which is involved in the benzenoid biosynthetic pathway and the production of BA. PhKAT1 is localised in the peroxisomes, where it is important for the formation of benzoyl-CoA-related compounds. Silencing of PhKAT1 resulted in a major reduction in BA and benzenoid formation, leaving the production of other phenylpropanoid-related volatiles unaffected. During the night, when volatile benzenoid production is highest, it is largely the beta-oxidative pathway that contributes to the formation of BA and benzenoids. Our studies add the benzenoid biosynthetic pathway to the list of pathways in which 3-ketoacyl-CoA thiolases are involved in plants.

Differentiation of long-chain fatty acid oxidation disorders using alternative precursors and acylcarnitine profiling in fibroblasts.

The differentiation of carnitine-acylcarnitine translocase deficiency (CACT) from carnitine palmitoyltransferase type II deficiency (CPT-II) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency from mitochondrial trifunctional protein deficiency (MTP) continues to be ambiguous using current acylcarnitine profiling techniques either from plasma or blood spots, or in the intact cell system (fibroblasts/amniocytes). Currently, enzyme assays are required to unequivocally differentiate CACT from CPT-II, and LCHAD from MTP. Over the years we have studied the responses of numerous FOD deficient cell lines to both even and odd numbered fatty acids of various chain lengths as well as branched-chain amino acids. In doing so, we discovered diagnostic elevations of unlabeled butyrylcarnitine detected only in CACT deficient cell lines when incubated with a shorter chain fatty acid, [7-2H3]heptanoate plus l-carnitine compared to the routinely used long-chain fatty acid, [16-2H3]palmitate. In monitoring the unlabeled C4/C5 acylcarnitine ratio, further differentiation from ETF/ETF-DH is also achieved. Similarly, incubating LCHAD and MTP deficient cell lines with the long-chain branched fatty acid, pristanic acid, and monitoring the C11/C9 acylcarnitine ratio has allowed differentiation between these disorders. These methods may be considered useful alternatives to specific enzyme assays for differentiation between these long-chain fatty acid oxidation disorders, as well as provide insight into new treatment strategies.

A teleost in vitro reporter gene assay to screen for agonists of the peroxisome proliferator-activated receptors.

Several contaminants detected in aquatic ecosystems are agonists of peroxisome proliferator-activated receptors (PPARs). Peroxisome proliferator-activated receptors interact with the retinoid X receptor (RXR) to activate the transcription of genes that control a variety of physiological functions. We cloned and sequenced partial cDNA fragments of rainbow trout (Oncorhynchus mykiss) PPARalpha and PPARbeta from rainbow trout (rt) gill-W1 cells, a cell line derived from rainbow trout gills; predicted amino acid identities are 77% and 82% compared with their respective human homologs and 83 to 88% and 91 to 98% identical to fish homologs. A reporter gene assay was developed by transfecting rt-gill-W1 cells with a reporter gene construct containing the peroxisome proliferator response element (PPRE) of the rat liver 3-ketoacyl-CoA thiolase B (TB) gene, which drives luciferase expression. Agonists of both PPARalpha (WY14,643 and gemfibrozil) and PPARbeta (bezafibrate) induced luciferase activity, while rosiglitazone, a PPARgamma agonist, was not effective. The fibrate drug, bezafibrate increased luciferase activity in a dose-dependent manner, but addition of 50 nM 9-cis-retinoic acid to the transfected rt-gill-W1 cell culture maximized the sensitivity of the assay so that bezafibrate could be detected at concentrations as low as 6 nM. Extracts from treated domestic wastewater containing fibrate drugs induced luciferase activity in the transfected gill cells. This in vitro reporter gene assay shows promise as a rapid and sensitive technique for screening environmental samples for PPAR-active substances.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency: T2-deficient patients with "mild" mutation(s) were previously misinterpreted as normal by the coupled assay with tiglyl-CoA.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of metabolism that affects the catabolism of isoleucine and ketone bodies. This disorder is characterized by intermittent ketoacidotic episodes. Recently, we diagnosed T2 deficiency in two patients (GK45 and GK47) by the absence of potassium ion-activated acetoacetyl-CoA thiolase activity, whereas these patients were previously misinterpreted as normal by a coupled assay with tiglyl-CoA as a substrate. This method has been widely used for the enzymatic diagnosis of the T2 deficiency in the United States and Europe. We hypothesized that some residual T2 activity showed normal results in the assay. To prove this hypothesis, we analyzed these two patients together with three typical T2-deficient patients (GK46, GK49, and GK50) at the DNA level. Expression analysis of mutant cDNAs clearly showed that GK45 and GK47 had "mild" mutations (A132G, D339-V340insD) that retained some residual T2 activity, at least one of two mutant alleles, whereas the other three patients had null mutations (c.52-53insC, G152A, H397D, and IVS8+1g>t) in either allele. These results raise the possibility that T2-deficient patients with mild mutations have been misinterpreted as normal by the coupled assay with tiglyl-CoA.

Constitutive expression of the beta-ketothiolase gene in transgenic plants. A major obstacle for obtaining polyhydroxybutyrate-producing plants.

Polyhydroxybutyrate (PHB) is a member of a class of thermoelastic polymers called polyhydroxyalkanoates that serve many bacteria as intracellular storage molecules for carbon and energy. Transgenic plants provide a potential means of producing this polymer cost-effectively. To date, however, few reports of the successful production of this polymer have been published, with the exception of work with transgenic Arabidopsis. Using a variety of chimeric constructs, we have determined that the constitutive, chloroplast-localized expression of one of the genes involved in PHB production-the beta-ketothiolase (phbA) gene-is detrimental to the efficient production of transgenic PHB. The alternate use of either inducible or somatically activated promoters allowed the construction of transgenic PHB-producing potato (Solanum tuberosum) and tobacco (Nicotiana tabacum) plants, although the amount of PHB formed was still rather low. Taking advantage of an inducible promoter, the maximal amount of PHB produced in transgenic potato was 0.09 mg g(-1) dry weight. In transgenic tobacco using a somatically activated promoter, up to 3.2 mg g(-1) dry weight was accumulated. In Arabidopsis, the formation of high levels of PHB had previously been shown to be accompanied by severe negative effects on growth and development of the plant. Phasins are proteins known from PHB-producing bacteria speculated to serve as protectants against the highly hydrophobic surface of the PHB granules in the bacterial intracellular milieu. Co-expression of the phasin gene in parallel with the PHB synthesis genes, however, did not lead to reduced symptom development.

Connection between poly-beta-hydroxybutyrate biosynthesis and growth on C(1) and C(2) compounds in the methylotroph Methylobacterium extorquens AM1.

Several DNA regions containing genes involved in poly-beta-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph Methylobacterium extorquens AM1. Genes involved in PHB biosynthesis include those encoding beta-ketothiolase (phaA), NADPH-linked acetoacetyl coenzyme A (acetyl-CoA) reductase (phaB), and PHB synthase (phaC). phaA and phaB are closely linked on the chromosome together with a third gene with identity to a regulator of PHB granule-associated protein, referred to as orf3. phaC was unlinked to phaA and phaB. Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (depA and depB). These genes show a high level of identity with each other at both DNA and amino acid levels. In addition, a gene encoding beta-hydroxybutyrate dehydrogenase (hbd) was identified. Insertion mutations were introduced into depA, depB, phaA, phaB, phaC, and hbd and also in a gene predicted to encode crotonase (croA), which is involved in fatty acid degradation, to investigate their role in PHB cycling. Mutants in depA, depB, hbd, and croA all produced normal levels of PHB, and the only growth phenotype observed was the inability of the hbd mutant to grow on beta-hydroxybutyrate. However, the phaA, phaB, and phaC mutants all showed defects in PHB synthesis. Surprisingly, these mutants also showed defects in growth on C(1) and C(2) compounds and, for phaB, these defects were rescued by glyoxylate supplementation. These results suggest that beta-hydroxybutyryl-CoA is an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate in methylotrophs and Streptomyces spp.

Comparative effects of perilla and fish oils on the activity and gene expression of fatty acid oxidation enzymes in rat liver.

The activity and mRNA level of hepatic enzymes in fatty acid oxidation and synthesis were compared in rats fed diets containing either 15% saturated fat (palm oil), safflower oil rich in linoleic acid, perilla oil rich in alpha-linolenic acid or fish oil rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) for 15 days. The mitochondrial fatty acid oxidation rate was 50% higher in rats fed perilla and fish oils than in the other groups. Perilla and fish oils compared to palm and safflower oils approximately doubled and more than tripled, respectively, peroxisomal fatty acid oxidation rate. Compared to palm and safflower oil, both perilla and fish oils caused a 50% increase in carnitine palmitoyltransferase I activity. Dietary fats rich in n-3 fatty acids also increased the activity of other fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. The extent of the increase was greater with fish oil than with perilla oil. Interestingly, both perilla and fish oils decreased the activity of 3-hydroxyacyl-CoA dehydrogenase measured using short- and medium-chain substrates. Compared to palm and safflower oils, perilla and fish oils increased the mRNA level of many mitochondrial and peroxisomal enzymes. Increases were generally greater with fish oil than with perilla oil. Fatty acid synthase, glucose-6-phosphate dehydrogenase, and pyruvate kinase activity and mRNA level were higher in rats fed palm oil than in the other groups. Among rats fed polyunsaturated fats, activities and mRNA levels of these enzymes were lower in rats fed fish oil than in the animals fed perilla and safflower oils. The values were comparable between the latter two groups. Safflower and fish oils but not perilla oil, compared to palm oil, also decreased malic enzyme activity and mRNA level. Examination of the fatty acid composition of hepatic phospholipid indicated that dietary alpha-linolenic acid is effectively desaturated and elongated to form EPA and DHA. Dietary perilla oil and fish oil therefore exert similar physiological activity in modulating hepatic fatty acid oxidation, but these dietary fats considerably differ in affecting fatty acid synthesis.

Identification and characterization of an intracellular protein complex that binds fibroblast growth factor-2 in bovine brain.

The fibroblast growth factor (FGF) family is composed of polypeptides with sequence identity which signal through transmembrane tyrosine kinase receptors. We report here the purification from bovine brain microsomes of an FGF-2-binding complex composed of three proteins of apparent molecular masses 150 kDa, 79 kDa and 46 kDa. Only the 150 kDa and 79 kDa proteins bound FGF-2 in cross-linking and ligand-blotting experiments. Binding of FGF-2 to p79 is enhanced in the presence of calcium. Peptide sequences allowed the identification of p150 and the cloning of the cDNAs encoding p79 and p46. The deduced amino acid sequence of p79 reveals high similarity to those of gastrin-binding protein and mitochondrial enoyl-CoA hydratase/hydroxyacyl-CoA dehydrogenase. p46 is similar to mitochondrial ketoacyl-CoA thiolase. Stable transfection of FR3T3 rat fibroblast cells with p79 cDNA analysed by electron microscopy following immunolabelling of ultra-thin cryosections revealed a localization of p79 in the secretory pathway, mainly in the endoplasmic reticulum and the Golgi region, where it is specifically associated with the molecular chaperone calnexin. In vivo a protein similar to the Golgi protein MG-160 forms a complex with FGF-2 and p79.

Sterol carrier protein X is peroxisomal 3-oxoacyl coenzyme A thiolase with intrinsic sterol carrier and lipid transfer activity.

Sterol carrier protein 2 (SCP2; also called nonspecific lipid transfer protein) is a small basic sterol carrier and lipid transfer protein assumed to participate in the intracellular transport of sterols and certain other lipids. Upon cloning and sequencing SCP2-encoding cDNAs, we and others found cDNAs containing unexpected in-frame 5'-extensions of up to 1,250 nucleotides upstream of the initiator ATG of the cDNA encoding pre-SCP2. The corresponding transcripts are primarily expressed in the liver and are predicted to encode a previously undescribed fusion protein containing a 143-amino acid C-terminal domain completely identical to pre-SCP2 and a 404-amino acid N-terminal domain with unknown biochemical activity or function (named sterol carrier protein x, SCPx). Here, we show that purified recombinant SCPx cleaves 3-oxoacyl(n)-CoA to yield acetyl-CoA and acyl(n-2)-CoA. Like SCP2, recombinant SCPx also stimulates the microsomal conversion of 7-dehydrocholesterol to cholesterol and transfers phosphatidylcholine and 7-dehydrocholesterol from small unilamellar vesicles to acceptor membranes in vitro. Furthermore, SCPx epitopes are primarily detected within peroxisomes. These findings suggest that SCPx is a previously undescribed peroxisomal 3-ketoacyl-CoA thiolase (EC 2.3.1.16) with intrinsic sterol carrier and lipid transfer activity (suggested name: SCP2/3-oxoacyl-CoA thiolase).

Molecular cloning of the cDNAs for the subunits of rat mitochondrial fatty acid beta-oxidation multienzyme complex. Structural and functional relationships to other mitochondrial and peroxisomal beta-oxidation enzymes.

Rat liver mitochondrial fatty acid oxidation multienzyme complex consists of 4 mol of the alpha-subunit and 4 mol of the beta-subunit, and has three enzyme activities of long chain enoyl-CoA hydratase, long chain 3-hydroxyacyl-CoA dehydrogenase, and long chain 3-ketoacyl-CoA thiolase. The following cDNA clones for the rat enzyme complex were isolated, sequenced, and expressed: 1) the 2,789-base pair (bp) cDNA clone had a 2,289-bp open reading frame encoding a 82,511-Da precursor and a 78,637-Da mature subunit. The deduced amino acid sequence of this subunit revealed that this cDNA encodes the alpha-subunit and had regions similar to the structure of rat mitochondrial enoyl-CoA hydratase and rat mitochondrial enoyl-CoA isomerase on the amino-terminal side, and a part similar to that of pig mitochondrial 3-hydroxyacyl-CoA dehydrogenase on the carboxyl-terminal side. Expression of this cDNA in COS-1 cells yielded a protein with long chain enoyl-CoA hydratase and long chain 3-hydroxyacyl-CoA dehydrogenase activities. 2) The 1,943-bp cDNA clone had a 1,425-bp open reading frame encoding a 51,413-Da precursor and a 47,583-Da mature subunit. A high similarity of the structure to 3-ketoacyl-CoA thiolases and acetoacetyl-CoA thiolases from various sources suggests that this clone encodes the beta-subunits. Expression of this cDNA in COS-1 cells yielded a protein with long chain 3-ketoacyl-CoA thiolase activity. By phylogenetic analysis of the deduced amino acid sequences of the alpha- and beta-subunits with those of other beta-oxidation enzymes, it was suggested that the alpha-subunit is a descendant of short chain enoyl-CoA hydratase and short chain 3-hydroxyacyl-CoA dehydrogenase while the beta-subunit first diverged from a common ancestor gene of the thiolase family.