High‐performance variants of plant diacylglycerol acyltransferase 1 generated by directed evolution provide insights into structure function

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the acyl‐CoA‐dependent biosynthesis of triacylglycerol, the predominant component of seed oil. In some oil crops, including Brassica napus, the level of DGAT1 activity can have a substantial effect on triacylglycerol production. Structure–function insights into DGAT1, however, remain limited because of the lack of a three‐dimensional detailed structure for this membrane‐bound enzyme. In this study, the amino acid residues governing B. napus DGAT1 (BnaDGAT1) activity were investigated via directed evolution, targeted mutagenesis, in vitro enzymatic assay, topological analysis, and transient expression of cDNA encoding selected enzyme variants in Nicotiana benthamiana. Directed evolution revealed that numerous amino acid residues were associated with increased BnaDGAT1 activity, and 67% of these residues were conserved among plant DGAT1s. The identified amino acid residue substitution sites occur throughout the BnaDGAT1 polypeptide, with 89% of the substitutions located outside the putative substrate binding or active sites. In addition, cDNAs encoding variants I447F or L441P were transiently overexpressed in N. benthamiana leaves, resulting in 33.2 or 70.5% higher triacylglycerol content, respectively, compared with native BnaDGAT1. Overall, the results provide novel insights into amino acid residues underlying plant DGAT1 function and performance‐enhanced BnaDGAT1 variants for increasing vegetable oil production.     

Chlamydomonas carries out fatty acid β‐oxidation in ancestral peroxisomes using a bona fide acyl‐CoA oxidase

Peroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002 (CrACX2), a gene encoding a member of the acyl‐CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl‐CoAs into trans‐2‐enoyl‐CoA and produced H₂O₂. This result demonstrated that CrACX2 is a genuine acyl‐CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) β‐oxidation spiral. A fluorescent protein‐tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA β‐oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of β‐oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant‐type FA β‐oxidation involving H₂O₂‐producing acyl‐CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii.     

A peroxisomal thioesterase plays auxiliary roles in plant β‐oxidative benzoic acid metabolism

Peroxisomal β‐oxidative degradation of compounds is a common metabolic process in eukaryotes. Reported benzoyl‐coenzyme A (BA‐CoA) thioesterase activity in peroxisomes from petunia flowers suggests that, like mammals and fungi, plants contain auxiliary enzymes mediating β‐oxidation. Here we report the identification of Petunia hybrida thioesterase 1 (PhTE1), which catalyzes the hydrolysis of aromatic acyl‐CoAs to their corresponding acids in peroxisomes. PhTE1 expression is spatially, developmentally and temporally regulated and exhibits a similar pattern to known benzenoid metabolic genes. PhTE1 activity is inhibited by free coenzyme A (CoA), indicating that PhTE1 is regulated by the peroxisomal CoA pool. PhTE1 downregulation in petunia flowers led to accumulation of BA‐CoA with increased production of benzylbenzoate and phenylethylbenzoate, two compounds which rely on the presence of BA‐CoA precursor in the cytoplasm, suggesting that acyl‐CoAs can be exported from peroxisomes. Furthermore, PhTE1 downregulation resulted in increased pools of cytoplasmic phenylpropanoid pathway intermediates, volatile phenylpropenes, lignin and anthocyanins. These results indicate that PhTE1 influences (i) intraperoxisomal acyl‐CoA/CoA levels needed to carry out β‐oxidation, (ii) efflux of β‐oxidative products, acyl‐CoAs and free acids, from peroxisomes, and (iii) flux distribution within the benzenoid/phenylpropanoid metabolic network. Thus, this demonstrates that plant thioesterases play multiple auxiliary roles in peroxisomal β‐oxidative metabolism.     

The structure of S. lividans acetoacetyl‐CoA synthetase shows a novel interaction between the C‐terminal extension and the N‐terminal domain

The adenosine monoposphate‐forming acyl‐CoA synthetase enzymes catalyze a two‐step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ～110 residue C‐terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl‐CoA synthetase (AacS) is presented that illustrates a novel aspect of this C‐terminal domain. Specifically, several acetyl‐ and acetoacetyl‐CoA synthetases contain a 30‐residue extension on the C‐terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N‐terminal domain. Proteins 2015; 83:575–581. © 2014 Wiley Periodicals, Inc.     

Arabidopsis acyl‐CoA‐binding protein ACBP1 participates in the regulation of seed germination and seedling development

A family of six genes encoding acyl‐CoA‐binding proteins (ACBPs), ACBP1–ACBP6, has been characterized in Arabidopsis thaliana. In this study, we demonstrate that ACBP1 promotes abscisic acid (ABA) signaling during germination and seedling development. ACBP1 was induced by ABA, and transgenic Arabidopsis ACBP1‐over‐expressors showed increased sensitivity to ABA during germination and seedling development, whereas the acbp1 mutant showed decreased ABA sensitivity during these processes. Subsequent RNA assays showed that ACBP1 over‐production in 12‐day‐old seedlings up‐regulated the expression of PHOSPHOLIPASE Dα1 (PLDα1) and three ABA/stress‐responsive genes: ABA‐RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1), RESPONSE TO DESICCATION29A (RD29A) and bHLH‐TRANSCRIPTION FACTOR MYC2 (MYC2). The expression of AREB1 and PLDα1 was suppressed in the acbp1 mutant in comparison with the wild type following ABA treatment. PLDα1 has been reported to promote ABA signal transduction by producing phosphatidic acid, an important lipid messenger in ABA signaling. Using lipid profiling, seeds and 12‐day‐old seedlings of ACBP1‐over‐expressing lines were shown to accumulate more phosphatidic acid after ABA treatment, in contrast to lower phosphatidic acid in the acbp1 mutant. Bimolecular fluorescence complementation assays indicated that ACBP1 interacts with PLDα1 at the plasma membrane. Their interaction was further confirmed by yeast two‐hybrid analysis. As recombinant ACBP1 binds phosphatidic acid and phosphatidylcholine, ACBP1 probably promotes PLDα1 action. Taken together, these results suggest that ACBP1 participates in ABA‐mediated seed germination and seedling development.     

Synthesis of oleyl oleate wax esters in Arabidopsis thaliana and Camelina sativa seed oil

Seed oil composed of wax esters with long‐chain monoenoic acyl moieties represents a high‐value commodity for industry. Such plant‐derived sperm oil‐like liquid wax esters are biodegradable and can have excellent properties for lubrication. In addition, wax ester oil may represent a superior substrate for biodiesel production. In this study, we demonstrate that the low‐input oil seed crop Camelina sativa can serve as a biotechnological platform for environmentally benign wax ester production. Two biosynthetic steps catalysed by a fatty alcohol‐forming acyl‐CoA reductase (FAR) and a wax ester synthase (WS) are sufficient to achieve wax ester accumulation from acyl‐CoA substrates. To produce plant‐derived sperm oil‐like liquid wax esters, the WS from Mus musculus (MmWS) or Simmondsia chinensis (ScWS) were expressed in combination with the FAR from Mus musculus (MmFAR1) or Marinobacter aquaeolei (MaFAR) in seeds of Arabidopsis thaliana and Camelina sativa. The three analysed enzyme combinations Oleo3:mCherry:MmFAR1?c/Oleo3:EYFP:MmWS, Oleo3:mCherry:MmFAR1?c/ScWS and MaFAR/ScWS showed differences in the wax ester molecular species profiles and overall biosynthetic performance. By expressing MaFAR/ScWS in Arabidopsis or Camelina up to 59% or 21% of the seed oil TAGs were replaced by wax esters, respectively. This combination also yielded wax ester molecular species with highest content of monounsaturated acyl moieties. Expression of the enzyme combinations in the Arabidopsis fae1 fad2 mutant background high in oleic acid resulted in wax ester accumulation enriched in oleyl oleate (18:1/18:1 > 60%), suggesting that similar values may be obtained with a Camelina high oleic acid line.     

ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT

Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.     

A NOVEL ASSAY OF ACYL‐COA:DIACYLGLYCEROL ACYLTRANSFERASE ACTIVITY UTILIZING FLUORESCENT SUBSTRATE1

Common methods for assaying acyl‐CoA:diacylglycerol acyltransferase (DGAT) enzymatic activity rely upon radiolabeled substrates or product assay. We developed a novel assay that directly quantifies endogenous DGAT activity through the use of a fluorescently labeled substrate. We performed this assay with microsomal protein, 2‐(6‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)hexanoyl‐1‐hexadecanoyl‐sn‐glycero‐3‐diacylglycerol (NBD‐DAG), and oleoyl‐CoA substrates. DGAT activity was analyzed in three species of algae as well as rat liver. The protocol proved to be sensitive and reliable. This assay may be used to facilitate research in the areas of biodiesel, oilseed crops, and triacylglycerol‐related human pathologies.     

Identification of a Chlamydomonas plastidial 2‐lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content

Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid‐targeted 2‐lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn‐2 position of a 2‐lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0‐CoA to 18:1‐CoA as an acyl donor. Fluorescent protein‐tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen‐deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid‐targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.     

Inhibitory effect of long chain fatty acyl CoAs on RNA polymerase from Escherichia coli

RNA polymerase from Escherichia coli was inhibited by long chain fatty acyl CoAs, such as myristoyl CoA (Ki = 17.2 microM), palmitoyl CoA (Ki = 8.9 microM), oleoyl CoA (Ki = 5.5 microM), and stearoyl CoA (Ki = 0.94 microM). The inhibition by these CoA thioesters was non-competitive against nucleoside triphosphates. Short chain fatty acyl CoAs, such as acetyl CoA, propionyl CoA, acetoacetyl CoA, butyryl CoA, and decanoyl CoA, failed to inhibit RNA polymerase. CoA, Na-myristate, Na-palmitate, Na-oleate, Na-stearate, palmitoyl carnitine, and carnitine did not inhibit the enzyme. The inhibition of RNA polymerase by long chain fatty acyl CoAs was competitive against template DNA.     

Rapid ester biosynthesis screening reveals a high activity alcohol‐O‐acyltransferase (AATase) from tomato fruit

Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl‐CoA with an alcohol by alcohol‐O‐acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short‐ and medium‐chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf‐S.l). Atf1‐S.l exhibited broad specificity towards acyl‐CoAs with chain length from C₄ to C₁₀ and was specific towards 1‐pentanol. The AATase screen also revealed new acyl‐CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf‐C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester‐based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.     

Overexpression of a Monocot Acyl‐CoA‐Binding Protein Confers Broad‐Spectrum Pathogen Protection in a Dicot

Plants are continuously infected by various pathogens throughout their lifecycle. Previous studies have reported that the expression of Class III acyl‐CoA‐binding proteins (ACBPs) such as the Arabidopsis ACBP3 and rice ACBP5 were induced by pathogen infection. Transgenic Arabidopsis AtACBP3‐overexpressors (AtACBP3‐OEs) displayed enhanced protection against the bacterial biotroph, Pseudomonas syringae, although they became susceptible to the fungal necrotroph Botrytis cinerea. A Class III ACBP from a monocot, rice (Oryza sativa) OsACBP5 was overexpressed in the dicot Arabidopsis. The resultant transgenic Arabidopsis lines conferred resistance not only to the bacterial biotroph P. syringae but to fungal necrotrophs (Rhizoctonia solani, B. cinerea, Alternaria brassicicola) and a hemibiotroph (Colletotrichum siamense). Changes in protein expression in R. solani‐infected Arabidopsis OsACBP5‐overexpressors (OsACBP5‐OEs) were demonstrated using proteomic analysis. Biotic stress‐related proteins including cell wall‐related proteins such as FASCILIN‐LIKE ARABINOGALACTAN‐PROTEIN10, LEUCINE‐RICH REPEAT EXTENSIN‐LIKE PROTEINS, XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE PROTEIN4, and PECTINESTERASE INHIBITOR18; proteins associated with glucosinolate degradation including GDSL‐LIKE LIPASE23, EPITHIOSPECIFIER MODIFIER1, MYROSINASE1, MYROSINASE2, and NITRILASE1; as well as a protein involved in jasmonate biosynthesis, ALLENE OXIDE CYCLASE2, were induced in OsACBP5‐OEs upon R. solani infection. These results indicated that upregulation of these proteins in OsACBP5‐OEs conferred protection against various plant pathogens.     

Vernonia DGATs increase accumulation of epoxy fatty acids in oil

Vernolic acid (cis‐12‐epoxy‐octadeca‐cis‐9‐enoic acid) is valuable as a renewable chemical feedstock. This fatty acid can accumulate to high levels in the seed oil of some plant species such as Vernonia galamensis and Stokesia laevis which are unsuitable for large‐scale production. A cost‐effective alternative for production of epoxy fatty acids is to genetically engineer its biosynthesis in commercial oilseeds. An epoxygenase cDNA (SlEPX) responsible for vernolic acid synthesis and two acyl‐CoA : diacylglycerol acyltransferase cDNAs (VgDGAT1 and VgDGAT2) catalysing triacylglycerol (TAG) formation were cloned from developing seeds of S. laevis and V. galamensis. Co‐expression of SlEPX and VgDGAT1 or VgDGAT2 greatly increases accumulation of vernolic acid both in petunia leaves and soybean somatic embryos. Seed‐specific expression of VgDGAT1 and VgDGAT2 in SlEPX mature soybean seeds results in vernolic acid levels of ～15% and 26%. Both DGAT1 and DGAT2 increase epoxy fatty acid accumulation with DGAT2 having much greater impact.     

Crystallographic and mass spectrometric analyses of a tandem GNAT protein from the clavulanic acid biosynthesis pathway

(3R,5R)‐Clavulanic acid (CA) is a clinically important inhibitor of Class A β‐lactamases. Sequence comparisons suggest that orf14 of the clavulanic acid biosynthesis gene cluster encodes for an acetyl transferase (CBG). Crystallographic studies reveal CBG to be a member of the emerging structural subfamily of tandem Gcn5‐related acetyl transferase (GNAT) proteins. Two crystal forms (C2 and P2₁ space groups) of CBG were obtained; in both forms one molecule of acetyl‐CoA (AcCoA) was bound to the N‐terminal GNAT domain, with the C‐terminal domain being unoccupied by a ligand. Mass spectrometric analyzes on CBG demonstrate that, in addition to one strongly bound AcCoA molecule, a second acyl‐CoA molecule can bind to CBG. Succinyl‐CoA and myristoyl‐CoA displayed the strongest binding to the “second” CoA binding site, which is likely in the C‐terminal GNAT domain. Analysis of the CBG structures, together with those of other tandem GNAT proteins, suggest that the AcCoA in the N‐terminal GNAT domain plays a structural role whereas the C‐terminal domain is more likely to be directly involved in acetyl transfer. The available crystallographic and mass spectrometric evidence suggests that binding of the second acyl‐CoA occurs preferentially to monomeric rather than dimeric CBG. The N‐terminal AcCoA binding site and the proposed C‐terminal acyl‐CoA binding site of CBG are compared with acyl‐CoA binding sites of other tandem and single domain GNAT proteins. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A novel role for central ACBP/DBI as a regulator of long‐chain fatty acid metabolism in astrocytes

Acyl‐CoA‐binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl‐CoA esters. Several studies have suggested that ACBP acts as an acyl‐CoA pool former and regulates long‐chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam‐Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABA_A receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism‐related gene expression using ACBP‐deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA‐CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes.