In vitro andin vivo biosynthesis of wax diesters by an unspecific bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase fromAcinetobacter calcoaceticus ADP1

2003 ◽  
Vol 105 (10) ◽  
pp. 578-584 ◽  
Author(s):  
Rainer Kalscheuer ◽  
Stefan Uthoff ◽  
Heinrich Luftmann ◽  
Alexander Steinbüchel
Keyword(s):  
Wax Ester ◽  
Wax Ester Synthase

scholarly journals Biosynthesis of Isoprenoid Wax Ester in Marinobacter hydrocarbonoclasticus DSM 8798: Identification and Characterization of Isoprenoid Coenzyme A Synthetase and Wax Ester Synthases

2007 ◽  
Vol 189 (10) ◽  
pp. 3804-3812 ◽  
Author(s):  
Erik Holtzapple ◽  
Claudia Schmidt-Dannert
Keyword(s):  
Coenzyme A ◽  
Draft Genome ◽  
Carbon Sources ◽  
Wax Ester ◽  
Sequence Information ◽  
Recombinant Enzymes ◽  
Available Carbon ◽  
Marinobacter Hydrocarbonoclasticus ◽  
Wax Ester Synthase

ABSTRACT Marinobacter hydrocarbonoclasticus DSM 8798 has been reported to synthesize isoprenoid wax ester storage compounds when grown on phytol as the sole carbon source under limiting nitrogen and/or phosphorous conditions. We hypothesized that isoprenoid wax ester synthesis involves (i) activation of an isoprenoid fatty acid by a coenzyme A (CoA) synthetase and (ii) ester bond formation between an isoprenoid alcohol and isoprenoyl-CoA catalyzed, most likely, by an isoprenoid wax ester synthase similar to an acyl wax ester synthase, wax ester synthase/diacylglycerol acyltransferase (WS/DGAT), recently described from Acinetobacter sp. strain ADP1. We used the recently released rough draft genome sequence of a closely related strain, M. aquaeolei VT8, to search for WS/DGAT and acyl-CoA synthetase candidate genes. The sequence information from putative WS/DGAT and acyl-CoA synthetase genes identified in this strain was used to clone homologues from the isoprenoid wax ester synthesizing Marinobacter strain. The activities of the recombinant enzymes were characterized, and two new isoprenoid wax ester synthases capable of synthesizing isoprenoid ester and acyl/isoprenoid hybrid ester in vitro were identified along with an isoprenoid-specific CoA synthetase. One of the Marinobacter wax ester synthases displays several orders of magnitude higher activity toward acyl substrates than any previously characterized acyl-WS and may reflect adaptations to available carbon sources in their environments.

scholarly journals Thio Wax Ester Biosynthesis Utilizing the Unspecific Bifunctional Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase of Acinetobacter sp. Strain ADP1

2005 ◽  
Vol 71 (2) ◽  
pp. 790-796 ◽  
Author(s):  
Stefan Uthoff ◽  
Tim Stöveken ◽  
Nikolaus Weber ◽  
Klaus Vosmann ◽  
Erika Klein ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Specific Activity ◽  
Wax Esters ◽  
Wax Ester ◽  
Mineral Salts ◽  
Relative Specific Activity ◽  
In Vitro Biosynthesis ◽  
Wax Ester Synthase

ABSTRACT The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter sp. strain ADP1 (formerly Acinetobacter calcoaceticus ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5′His6WS/DGAT comprising an N-terminal His6 tag was purified from the soluble protein fraction of Escherichia coli Rosetta(DE3)pLysS (pET23a::5′His6 atf). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein−1 min−1 was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5′His6WS/DGAT with 1-hexadecanethiol and palmitoyl-CoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-S-monopalmitoyloctanedithiol and minor amounts of 1,8-S-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-S-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant Acinetobacter sp. strain ADP1acr1ΩKm, which is unable to produce fatty alcohols, was used. Cultivation of Acinetobacter sp. strain ADP1acr1ΩKm in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry.

scholarly journals Do minke whales (Balaenoptera acutorostrata) digest wax esters?

1995 ◽  
Vol 74 (5) ◽  
pp. 717-722 ◽  
Author(s):  
Erling S. Nordøy
Keyword(s):  
Energy Density ◽  
Dry Matter ◽  
General Pattern ◽  
Dry Mass ◽  
Wax Esters ◽  
Gut Contents ◽  
Wax Ester ◽  
Balaenoptera Acutorostrata ◽  
Minke Whales

Mammals are known to utilize wax esters with an efficiency of less than 50%. The purpose of the present study was to examine whether or not minke whales (Balaenoptera acutorostrata), which at times may eat considerable amounts of wax-ester-rich krill, represent an exception to this general pattern. Samples of fresh undigested forestomach, as well as colon, contents were obtained from minke whales (n5) that had been feeding on krill (Thysanoessa inermis) for some time. The samples were analysed for dry mass, energy density, lipid content and the major lipid classes, including wax esters. The concentrations of wax esters were compared with previous estimates of dry-matter disappearance of the same type of prey using anin vitrotechnique, to calculate the dry-matter digestibility of wax esters (DMDwax). Wax esters contributed 21% of the energy and 47% of total lipids in the krill diet. The energy density of gut contents decreased by 50% after their passage from forestomach to the end of the colon. The DMDwaxwas 94·1 (SD 2·8)% (n5). This high DMDwaxand the occurrence of fatty alcohols, one of the products of wax-ester hydrolysis, in faeces show that minke whales are very efficient digesters of wax esters and absorb most of the energy-rich products of this process.

scholarly journals Wax Ester Synthesis is Required for Mycobacterium tuberculosis to Enter In Vitro Dormancy

PLoS ONE ◽  
2012 ◽  
Vol 7 (12) ◽  
pp. e51641 ◽  
Author(s):  
Tatiana D. Sirakova ◽  
Chirajyoti Deb ◽  
Jaiyanth Daniel ◽  
Harminder D. Singh ◽  
Hedia Maamar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Wax Ester ◽  
Ester Synthesis

Directed evolution of a bacterial WS/DGAT acyltransferase: improving tDGAT from Thermomonospora curvata

2019 ◽  
Author(s):  
Omar Santín ◽  
Serena Galié ◽  
Gabriel Moncalián
Keyword(s):  
Directed Evolution ◽  
High Throughput Screening ◽  
Neutral Lipids ◽  
Nile Red ◽  
Diacylglycerol Acyltransferase ◽  
Wax Ester ◽  
Bacterial Lipid ◽  
Acyl Coenzyme A ◽  
Wax Ester Synthase ◽  
Tag Accumulation

Abstract Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.

scholarly journals Fatty Alcohols for Wax Esters in Marinobacter aquaeolei VT8: Two Optional Routes in the Wax Biosynthesis Pathway

2013 ◽  
Vol 79 (22) ◽  
pp. 7055-7062 ◽  
Author(s):  
Eric M. Lenneman ◽  
Janet M. Ohlert ◽  
Nagendra P. Palani ◽  
Brett M. Barney
Keyword(s):  
Fatty Alcohol ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Transcriptional Analysis ◽  
Wax Ester ◽  
Fatty Aldehyde ◽  
Marinobacter Aquaeolei

ABSTRACTThe biosynthesis of wax esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previousin vitroenzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol inMarinobacter aquaeoleiVT8. In this study, we demonstrate through a series of gene deletions and transcriptional analysis that either enzyme is capable of fulfilling the role of providing the fatty alcohol required for wax ester biosynthesisin vivo, but evolution has clearly selected one of these, a previously characterized fatty aldehyde reductase, as the preferred enzyme to perform this reaction under typical wax ester-accumulating conditions. These results complement previousin vitrostudies and provide the first glimpse into the role of each enzymein vivoin the native organism.

scholarly journals Multiple Pathways for Triacylglycerol Biosynthesis in Streptomyces coelicolor

2008 ◽  
Vol 74 (9) ◽  
pp. 2573-2582 ◽  
Author(s):  
Ana Arabolaza ◽  
Eduardo Rodriguez ◽  
Silvia Altabe ◽  
Hector Alvarez ◽  
Hugo Gramajo
Keyword(s):  
Mutant Strain ◽  
De Novo ◽  
Streptomyces Coelicolor ◽  
Lipid Production ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Triple Mutant ◽  
Storage Lipids ◽  
Tag Biosynthesis

ABSTRACT The terminal reaction in triacylglyceride (TAG) biosynthesis is the esterification of diacylglycerol (DAG) with a fatty acid molecule. To study this reaction in Streptomyces coelicolor, we analyzed three candidate genes (sco0958, sco1280, and sco0123) whose products significantly resemble the recently identified wax ester synthase/acyl-coenzyme A (CoA):DAG acyltransferase (DGAT) from Acinetobacter baylyi. The deletion of either sco0123 or sco1280 resulted in no detectable decrease in TAG accumulation. In contrast, the deletion of sco0958 produced a dramatic reduction in neutral lipid production, whereas the overexpression of this gene yielded a significant increase in de novo TAG biosynthesis. In vitro activity assays showed that Sco0958 mediates the esterification of DAG using long-chain acyl-CoAs (C14 to C18) as acyl donors. The Km and V max values of this enzyme for myristoyl-CoA were 45 μM and 822 nmol mg−1 min−1, respectively. Significantly, the triple mutant strain was not completely devoid of storage lipids, indicating the existence of alternative TAG-biosynthetic routes. We present strong evidence demonstrating that the residual production of TAG in this mutant strain is mediated, at least in part, by an acyl-CoA-dependent pathway, since the triple mutant still exhibited DGAT activity. More importantly, there was substantial phospholipid:DGAT (PDAT) activity in the wild type and in the triple mutant. This is the first time that a PDAT activity has been reported for bacteria, highlighting the extreme metabolic diversity of this industrially important soil microorganism.

scholarly journals Role of Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase in Oleaginous Streptomyces sp. Strain G25

2016 ◽  
Vol 82 (19) ◽  
pp. 5969-5981 ◽  
Author(s):  
Annika Röttig ◽  
Carl Simon Strittmatter ◽  
Jennifer Schauer ◽  
Sebastian Hiessl ◽  
Anja Poehlein ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Genome Sequence ◽  
Lipid Accumulation ◽  
Draft Genome ◽  
Dry Weight ◽  
Lipid Storage ◽  
Draft Genome Sequence ◽  
Wax Ester ◽  
Content Type ◽  
Wax Ester Synthase

ABSTRACTRecently, we isolated a novelStreptomycesstrain which can accumulate extraordinarily large amounts of triacylglycerol (TAG) and consists of 64% fatty acids (dry weight) when cultivated with glucose and 50% fatty acids (dry weight) when cultivated with cellobiose. To identify putative gene products responsible for lipid storage and cellobiose utilization, we analyzed its draft genome sequence. A single gene encoding a wax ester synthase/acyl coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) was identified and heterologously expressed inEscherichia coli. The purified enzyme AtfG25showed acyltransferase activity with C12- or C16-acyl-CoA, C12to C18alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA fromAcinetobacter baylyibut has high sequence similarities to WS/DGATs from otherStreptomycesspecies. To investigate the impact of AtfG25on lipid accumulation, the respective gene,atfG25, was inactivated inStreptomycessp. strain G25. However, cells of the insertion mutant still exhibited DGAT activity and were able to store TAG, albeit in lower quantities and at lower rates than the wild-type strain. These findings clearly indicate that AtfG25has an important, but not exclusive, role in TAG biosynthesis in the novelStreptomycesisolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study.IMPORTANCEA novelStreptomycesstrain was isolated from desert soil, which represents an extreme environment with high temperatures, frequent drought, and nutrient scarcity. We believe that these harsh conditions promoted the development of the capacity for this strain to accumulate extraordinarily large amounts of lipids. In this study, we present the analysis of its draft genome sequence with a special focus on enzymes potentially involved in its lipid storage. Furthermore, the activity and importance of the detected acyltransferase were studied. As discussed in this paper, and in contrast to many other bacteria, streptomycetes seem to possess a complex metabolic network to synthesize lipids, whereof crucial steps are still largely unknown. This paper therefore provides insights into a range of topics, including extremophile bacteria, the physiology of lipid accumulation, and the biotechnological production of bacterial lipids.

scholarly journals The Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase from Acinetobacter sp. Strain ADP1: Characterization of a Novel Type of Acyltransferase

2005 ◽  
Vol 187 (4) ◽  
pp. 1369-1376 ◽  
Author(s):  
Tim Stöveken ◽  
Rainer Kalscheuer ◽  
Ursula Malkus ◽  
Rudolf Reichelt ◽  
Alexander Steinbüchel
Keyword(s):  
Wax Ester ◽  
Biotechnological Production ◽  
Medium Chain Length ◽  
Homologous Genes ◽  
Apparent Homogeneity ◽  
Acyl Coenzyme A ◽  
Wax Ester Synthase ◽  
Purification Protocol

ABSTRACT The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium Acinetobacter sp. strain ADP1. It constitutes a novel class of acyltransferases which is fundamentally different from acyltransferases involved in TAG and WE synthesis in eukaryotes. The enzyme was purified by a three-step purification protocol to apparent homogeneity from the soluble fraction of recombinant Escherichia coli Rosetta (DE3)pLysS (pET23a::atfA). Purified WS/DGAT revealed a remarkably low substrate specificity, accepting a broad range of various substances as alternative acceptor molecules. Besides having DGAT and WS activity, the enzyme possesses acyl-CoA:monoacylglycerol acyltransferase (MGAT) activity. The sn-1 and sn-3 positions of acylglycerols are accepted with higher specificity than the sn-2 position. Linear alcohols ranging from ethanol to triacontanol are efficiently acylated by the enzyme, which exhibits highest specificities towards medium-chain-length alcohols. The acylation of cyclic and aromatic alcohols, such as cyclohexanol or phenylethanol, further underlines the unspecific character of this enzyme. The broad range of possible substrates may lead to biotechnological production of interesting wax ester derivatives. Determination of the native molecular weight revealed organization as a homodimer. The large number of WS/DGAT-homologous genes identified in pathogenic mycobacteria and their possible importance for the pathogenesis and latency of these bacteria makes the purified WS/DGAT from Acinetobacter sp. strain ADP1 a valuable model for studying this group of proteins in pathogenic mycobacteria.

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