Correction: Prasad et al. Patterns of Variation in the Usage of Fatty Acid Chains among Classes of Ester and Ether Neutral Lipids and Phospholipids in the Queensland Fruit Fly. Insects 2023, 14, 873.

Supplementary Table S3 in our recent publication [...].

Patterns of Variation in the Usage of Fatty Acid Chains among Classes of Ester and Ether Neutral Lipids and Phospholipids in the Queensland Fruit Fly.

Modern lipidomics has the power and sensitivity to elucidate the role of insects' lipidomes in their adaptations to the environment at a mechanistic molecular level. However, few lipidomic studies have yet been conducted on insects beyond model species such as Drosophila melanogaster. Here, we present the lipidome of adult males of another higher dipteran frugivore, Bactrocera tryoni. We describe 421 lipids across 15 classes of ester neutral lipids and phospholipids and ether neutral lipids and phospholipids. Most of the lipids are specified in terms of the carbon and double bond contents of each constituent hydrocarbon chain, and more ether lipids are specified to this degree than in any previous insect lipidomic analyses. Class-specific profiles of chain length and (un)saturation are broadly similar to those reported in D. melanogaster, although we found fewer medium-length chains in ether lipids. The high level of chain specification in our dataset also revealed widespread non-random combinations of different chain types in several ester lipid classes, including deficits of combinations involving chains of the same carbon and double bond contents among four phospholipid classes and excesses of combinations of dissimilar chains in several classes. Large differences were also found in the length and double bond profiles of the acyl vs. alkyl or alkenyl chains of the ether lipids. Work on other organisms suggests some of the differences observed will be functionally consequential and mediated, at least in part, by differences in substrate specificity among enzymes in lipid synthesis and remodelling pathways. Interrogation of the B. tryoni genome showed it has comparable levels of diversity overall in these enzymes but with some gene gain/loss differences and considerable sequence divergence from D. melanogaster.

Recombinant Aflatoxin-Degrading F420H2-Dependent Reductase from Mycobacterium smegmatis Protects Mammalian Cells from Aflatoxin Toxicity.

Aflatoxins are carcinogenic secondary metabolites of fungi that contaminate many staple crops and foods. Aflatoxin contamination is a worldwide problem, especially in developing countries, posing health hazards, e.g., causing aflatoxicosis and hepatocellular carcinoma, and even death. Biological solutions for aflatoxin detoxification are environmentally friendly and a cheaper alternative than chemical methods. The aims of the current study were to investigate: (1) the ability of MSMEG_5998, an aflatoxin-degrading F420H2-dependent reductase from Mycobacterium smegmatis, to degrade aflatoxin B1 (AFB1) and reduce AFB1-caused damage in HepG2 cell culture model; and (2) whether a thioredoxin (Trx) linkage of MSMEG_5998 enhanced the enzyme activity. We show that Trx-linked MSMEG_5998 degraded 63% AFB1 and native MSMEG_5998 degraded 31% after 4 h at 22 °C, indicating that the Trx-linked enzyme had a better AFB1-degrading ability. In a HepG2 cell culture model, Trx-linked MSMEG_5998 reduced DNA damage and p53-mediated apoptosis caused by AFB1 to a greater extent than the native enzyme. These findings suggest that Trx-linked MSMEG_5998 could potentially be developed to protect the liver from AFB1 damage, or as a candidate protein to reduce AFB1-related toxicity in animals.

Up-regulation of lipid biosynthesis increases the oil content in leaves of Sorghum bicolor.

Synthesis and accumulation of the storage lipid triacylglycerol in vegetative plant tissues has emerged as a promising strategy to meet the world's future need for vegetable oil. Sorghum (Sorghum bicolor) is a particularly attractive target crop given its high biomass, drought resistance and C4 photosynthesis. While oilseed-like triacylglycerol levels have been engineered in the C3 model plant tobacco, progress in C4 monocot crops has been lagging behind. In this study, we report the accumulation of triacylglycerol in sorghum leaf tissues to levels between 3 and 8.4% on a dry weight basis depending on leaf and plant developmental stage. This was achieved by the combined overexpression of genes encoding the Zea mays WRI1 transcription factor, Umbelopsis ramanniana UrDGAT2a acyltransferase and Sesamum indicum Oleosin-L oil body protein. Increased oil content was visible as lipid droplets, primarily in the leaf mesophyll cells. A comparison between a constitutive and mesophyll-specific promoter driving WRI1 expression revealed distinct changes in the overall leaf lipidome as well as transitory starch and soluble sugar levels. Metabolome profiling uncovered changes in the abundance of various amino acids and dicarboxylic acids. The results presented here are a first step forward towards the development of sorghum as a dedicated biomass oil crop and provide a basis for further combinatorial metabolic engineering.

Increased DHA Production in Seed Oil Using a Selective Lysophosphatidic Acid Acyltransferase.

Metabolic engineering of the omega-3 (ω3) long chain polyunsaturated fatty acid biosynthesis pathway has generated fish oil-like levels of pharmaceutically and nutritionally important docosahexaenoic acid (DHA) in plant seeds. However, the majority of DHA has been accumulated at the sn-1 and sn-3 positions of triacylglycerol (TAG) in these engineered seeds, leaving only a minor amount (∼10%) at sn-2 position and indicating a strong discrimination (or, a very poor specificity) for DHA by seed lysophosphatidic acid acyltransferases (LPAATs), which mediate the acylation of sn-2 position of glycerol backbone. In order to increase the level of DHA at sn-2 position of TAG and to increase overall DHA level in seeds, we attempted to discover DHA-preferring LPAATs. Several LPAATs for acylation of the sn-2 position of the TAG glycerol backbone were investigated for substrate preference for DHA. In transiently expressing these LPAATs in Nicotiana benthamiana, a Mortierella alpina LPAAT had the highest substrate specificity for accumulating DHA onto oleoyl-lysophosphatidic acid (oleoyl-LPA), while the plant LPAATs tested showed lower preference for DHA. In a competition assay with a pool of four ω3 acyl-Coenzyme A (CoA) substrates involved in the DHA biosynthesis pathway, LPAATs from both M. alpina and Emiliania huxleyi showed a high preference for DHA-CoA acylation onto oleoyl-LPA. When docosahexaenoyl-LPA was used as the acyl receiver, M. alpina LPAAT also showed a high preference for DHA-CoA. Stable overexpression of M. alpina LPAAT in an Arabidopsis line that expressed the DHA biosynthesis pathway significantly increased both the total DHA levels and the distribution of DHA onto the sn-2 position of seed TAG. LC-MS analysis of the seed TAG species also confirmed that overexpression of M. alpina LPAAT increased di-DHA and tri-DHA TAGs, suggesting that the M. alpina LPAAT could enrich DHA at the TAG sn-2 position, leading to a metabolic engineering of oil seed for channeling DHA into the sn-2 position of TAG and to a higher DHA level.

Seed-specific RNAi in safflower generates a superhigh oleic oil with extended oxidative stability.

Vegetable oils extracted from oilseeds are an important component of foods, but are also used in a range of high value oleochemical applications. Despite being biodegradable, nontoxic and renewable current plant oils suffer from the presence of residual polyunsaturated fatty acids that are prone to free radical formation that limit their oxidative stability, and consequently shelf life and functionality. Many decades of plant breeding have been successful in raising the oleic content to ~90%, but have come at the expense of overall field performance, including poor yields. Here, we engineer superhigh oleic (SHO) safflower producing a seed oil with 93% oleic generated from seed produced in multisite field trials spanning five generations. SHO safflower oil is the result of seed-specific hairpin-based RNA interference of two safflower lipid biosynthetic genes, FAD2.2 and FATB, producing seed oil containing less than 1.5% polyunsaturates and only 4% saturates but with no impact on lipid profiles of leaves and roots. Transgenic SHO events were compared to non-GM safflower in multisite trial plots with a wide range of growing season conditions, which showed no evidence of impact on seed yield. The oxidative stability of the field-grown SHO oil produced from various sites was 50 h at 110°C compared to 13 h for conventional ~80% oleic safflower oils. SHO safflower produces a uniquely stable vegetable oil across different field conditions that can provide the scale of production that is required for meeting the global demands for high stability oils in food and the oleochemical industry.

A reconfigured Kennedy pathway which promotes efficient accumulation of medium-chain fatty acids in leaf oils.

Medium-chain fatty acids (MCFA, C6-14 fatty acids) are an ideal feedstock for biodiesel and broader oleochemicals. In recent decades, several studies have used transgenic engineering to produce MCFA in seeds oils, although these modifications result in unbalance membrane lipid profiles that impair oil yields and agronomic performance. Given the ability to engineer nonseed organs to produce oils, we have previously demonstrated that MCFA profiles can be produced in leaves, but this also results in unbalanced membrane lipid profiles and undesirable chlorosis and cell death. Here we demonstrate that the introduction of a diacylglycerol acyltransferase from oil palm, EgDGAT1, was necessary to channel nascent MCFA directly into leaf oils and therefore bypassing MCFA residing in membrane lipids. This pathway resulted in increased flux towards MCFA rich leaf oils, reduced MCFA in leaf membrane lipids and, crucially, the alleviation of chlorosis. Deep sequencing of African oil palm (Elaeis guineensis) and coconut palm (Cocos nucifera) generated candidate genes of interest, which were then tested for their ability to improve oil accumulation. Thioesterases were explored for the production of lauric acid (C12:0) and myristic (C14:0). The thioesterases from Umbellularia californica and Cinnamomum camphora produced a total of 52% C12:0 and 40% C14:0, respectively, in transient leaf assays. This study demonstrated that the introduction of a complete acyl-CoA-dependent pathway for the synthesis of MFCA-rich oils avoided disturbing membrane homoeostasis and cell death phenotypes. This study outlines a transgenic strategy for the engineering of biomass crops with high levels of MCFA rich leaf oils.

Step changes in leaf oil accumulation via iterative metabolic engineering.

Synthesis and accumulation of plant oils in the entire vegetative biomass offers the potential to deliver yields surpassing those of oilseed crops. However, current levels still fall well short of those typically found in oilseeds. Here we show how transcriptome and biochemical analyses pointed to a futile cycle in a previously established Nicotiana tabacum line, accumulating up to 15% (dry weight) of the storage lipid triacylglycerol in leaf tissue. To overcome this metabolic bottleneck, we either silenced the SDP1 lipase or overexpressed the Arabidopsis thaliana LEC2 transcription factor in this transgenic background. Both strategies independently resulted in the accumulation of 30-33% triacylglycerol in leaf tissues. Our results demonstrate that the combined optimization of de novo fatty acid biosynthesis, storage lipid assembly and lipid turnover in leaf tissue results in a major overhaul of the plant central carbon allocation and lipid metabolism. The resulting further step changes in oil accumulation in the entire plant biomass offers the possibility of delivering yields that outperform current oilseed crops.

Persistence of the dominant soil phylum Acidobacteria by trace gas scavenging.

The majority of microbial cells in global soils exist in a spectrum of dormant states. However, the metabolic processes that enable them to survive environmental challenges, such as nutrient-limitation, remain to be elucidated. In this work, we demonstrate that energy-starved cultures of Pyrinomonas methylaliphatogenes, an aerobic heterotrophic acidobacterium isolated from New Zealand volcanic soils, persist by scavenging the picomolar concentrations of H2 distributed throughout the atmosphere. Following the transition from exponential to stationary phase due to glucose limitation, the bacterium up-regulates by fourfold the expression of an eight-gene operon encoding an actinobacteria-type H2-uptake [NiFe]-hydrogenase. Whole-cells of the organism consume atmospheric H2 in a first-order kinetic process. Hydrogen oxidation occurred most rapidly under oxic conditions and was weakly associated with the cell membrane. We propose that atmospheric H2 scavenging serves as a mechanism to sustain the respiratory chain of P. methylaliphatogenes when organic electron donors are scarce. As the first observation of H2 oxidation to our knowledge in the Acidobacteria, the second most dominant soil phylum, this work identifies new sinks in the biogeochemical H2 cycle and suggests that trace gas oxidation may be a general mechanism for microbial persistence.

Metabolic engineering of medium-chain fatty acid biosynthesis in Nicotiana benthamiana plant leaf lipids.

Various research groups are investigating the production of oil in non-seed biomass such as leaves. Recently, high levels of oil accumulation have been achieved in plant biomass using a combination of biotechnological approaches which also resulted in significant changes to the fatty acid composition of the leaf oil. In this study, we were interested to determine whether medium-chain fatty acids (MCFA) could be accumulated in leaf oil. MCFA are an ideal feedstock for biodiesel and a range of oleochemical products including lubricants, coatings, and detergents. In this study, we explore the synthesis, accumulation, and glycerolipid head-group distribution of MCFA in leaves of Nicotiana benthamiana after transient transgenic expression of C12:0-, C14:0-, and C16:0-ACP thioesterase genes. We demonstrate that the production of these MCFA in leaf is increased by the co-expression of the WRINKLED1 (WRI1) transcription factor, with the lysophosphatidic acid acyltransferase (LPAAT) from Cocos nucifera being required for the assembly of tri-MCFA TAG species. We also demonstrate that the newly-produced MCFA are incorporated into the triacylglycerol of leaves in which WRI1 + diacylglycerol acyltransferase1 (DGAT1) genes are co-expressed for increased oil accumulation.

Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: purification, activity, and binding properties toward multiple pesticides.

Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmol L⁻¹ of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC50 values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects.

Deubiquitylating enzymes and disease.

Deubiquitylating enzymes (DUBs) can hydrolyze a peptide, amide, ester or thiolester bond at the C-terminus of UBIQ (ubiquitin), including the post-translationally formed branched peptide bonds in mono- or multi-ubiquitylated conjugates. DUBs thus have the potential to regulate any UBIQ-mediated cellular process, the two best characterized being proteolysis and protein trafficking. Mammals contain some 80-90 DUBs in five different subfamilies, only a handful of which have been characterized with respect to the proteins that they interact with and deubiquitylate. Several other DUBs have been implicated in various disease processes in which they are changed by mutation, have altered expression levels, and/or form part of regulatory complexes. Specific examples of DUB involvement in various diseases are presented. While no specific drugs targeting DUBs have yet been described, sufficient functional and structural information has accumulated in some cases to allow their rapid development. PUBLICATION HISTORY : Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

Deficiency of glutathione transferase zeta causes oxidative stress and activation of antioxidant response pathways.

Glutathione S-transferase (GST) zeta (GSTZ1-1) plays a significant role in the catabolism of phenylalanine and tyrosine, and a deficiency of GSTZ1-1 results in the accumulation of maleylacetoacetate and its derivatives maleylacetone (MA) and succinylacetone. Induction of GST subunits was detected in the liver of Gstz1(-/-) mice by Western blotting with specific antisera and high-performance liquid chromatography analysis of glutathione affinity column-purified proteins. The greatest induction was observed in members of the mu class. Induction of NAD(P)H:quinone oxidoreductase 1 and the catalytic and modifier subunits of glutamate-cysteine ligase was also observed. Many of the enzymes that are induced in Gstz1(-/-) mice are regulated by antioxidant response elements that respond to oxidative stress via the Keap1/Nrf2 pathway. It is significant that diminished glutathione concentrations were also observed in the liver of Gstz1(-/-) mice, which supports the conclusion that under normal dietary conditions, the accumulation of electrophilic intermediates such as maleylacetoacetate and MA results in a high level of oxidative stress. Elevated GST activities in the livers of Gstz1(-/-) mice suggest that GSTZ1-1 deficiency may alter the metabolism of some drugs and xenobiotics. Gstz1(-/-) mice given acetaminophen demonstrated increased hepatotoxicity compared with wild-type mice. This toxicity may be attributed to the increased GST activity or the decreased hepatic concentrations of glutathione, or both. Patients with acquired deficiency of GSTZ1-1 caused by therapeutic exposure to dichloroacetic acid for the clinical treatment of lactic acidosis may be at increased risk of drug- and chemical-induced toxicity.

Ultrastructural changes in hepatic sinusoidal endothelial cells acutely exposed to colloidal iron.

Hepatic sinusoidal endothelial cells form an important interface between the vascular system, represented by the sinusoids, and the space of Disse that surrounds the hepatocyte microvilli. This study aimed to assess the light microscopic and ultrastructural effects of acute exposure of hepatic sinusoidal endothelial cells to colloidal iron by injection of rats with iron polymaltose. Eight minutes after a single intravenous injection of iron polymaltose sinusoidal endothelial cells showed defenestration, and thickening and layering as assessed by transmission electron microscopy. Kupffer cells and stellate cells appeared activated. These changes were not observed in control animals, experiments using equivalent doses of maltose, or experiments using colloidal carbon except for Kupffer cell activation due to colloidal carbon. No significant light microscopic changes were seen in study or control animals. The findings indicate that acute exposure to colloidal iron causes changes in hepatic sinusoidal endothelial cells, stellate cells and Kupffer cells. This may be the result of a direct toxic effect of iron or increased production of reactive oxygen species. These observations suggest a possible mechanism for defenestration of sinusoidal endothelial cells in ageing and in disease states.

Clarification of the role of key active site residues of glutathione transferase zeta/maleylacetoacetate isomerase by a new spectrophotometric technique.

hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(+/-)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16-->Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the K(m) for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of alpha-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the alpha-halo acid substrates.