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Alexander Steinbuchel - One of the best experts on this subject based on the ideXlab platform.
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role of Wax Ester synthase acyl coenzyme a diacylglycerol acyltransferase in oleaginous streptomyces sp strain g25
Applied and Environmental Microbiology, 2016Co-Authors: Annika Rottig, Alexander Steinbuchel, Carl Simon Strittmatter, Jennifer Schauer, Sebastian Hiessl, Anja Poehlein, Rolf DanielAbstract:ABSTRACT Recently, we isolated a novel Streptomyces strain 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 in Escherichia coli. The purified enzyme AtfG25 showed acyltransferase activity with C12- or C16-acyl-CoA, C12 to C18 alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA from Acinetobacter baylyi but has high sequence similarities to WS/DGATs from other Streptomyces species. To investigate the impact of AtfG25 on lipid accumulation, the respective gene, atfG25, was inactivated in Streptomyces sp. 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 AtfG25 has an important, but not exclusive, role in TAG biosynthesis in the novel Streptomyces isolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study. IMPORTANCE A novel Streptomyces strain 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.
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both histidine residues of the conserved hhxxxdg motif are essential for Wax Ester synthase acyl coa diacylglycerol acyltransferase catalysis
European Journal of Lipid Science and Technology, 2009Co-Authors: Tim Stoveken, Rainer Kalscheuer, Alexander SteinbuchelAbstract:Bacterial acyltransferases of the Wax Ester synthase/diacylglycerol acyltransferase (WS/DGAT) family possess a highly conserved HHXXXDG motif. In this study, we describe the first experimental evidence that this motif is part of the active site of WS/DGAT from the Acinetobacter baylyi strain ADP1 and that it is crucial for enzymatic activity. The second histidine residue of this motif (H133) turned out to be essential for the catalytic activity. In addition, the replacement of the first histidine (His132) also led to explicitly decreased activity. A complete loss of activity was only observed upon substitution of both histidine residues by leucine, revealing that both are necessary for maximal activity. In contrast, the replacement of Asp137 and Gly138 against alanine had only little effect on enzyme activity, thus demonstrating that they are not essential for WS/DGAT catalysis although belonging to the highly conserved motif. One peculiarity of WS/DGAT enzymes is their little substrate specificity regarding hydrophobic compounds. In this study, we demonstrated the inability of WS/DGAT to accept polar compounds as substrates.
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the Wax Ester synthase acyl coenzyme a diacylglycerol acyltransferase from acinetobacter sp strain adp1 characterization of a novel type of acyltransferase
Journal of Bacteriology, 2005Co-Authors: Tim Stoveken, Rainer Kalscheuer, Ursula Malkus, Rudolf Reichelt, Alexander SteinbuchelAbstract: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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thio Wax Ester biosynthesis utilizing the unspecific bifunctional Wax Ester synthase acyl coenzyme a diacylglycerol acyltransferase of acinetobacter sp strain adp1
Applied and Environmental Microbiology, 2005Co-Authors: Stefan Uthoff, Tim Stoveken, N Weber, Klaus Vosmann, Erika Klein, Rainer Kalscheuer, Alexander SteinbuchelAbstract: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′His6atf). 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.
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in vitro and in vivo biosynthesis of Wax diEsters by an unspecific bifunctional Wax Ester synthase acyl coa diacylglycerol acyltransferase from acinetobacter calcoaceticus adp1
European Journal of Lipid Science and Technology, 2003Co-Authors: Rainer Kalscheuer, Stefan Uthoff, Heinrich Luftmann, Alexander SteinbuchelAbstract:Acinetobacter calcoaceticus ADP1 possesses a bifunctional Wax Ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) catalyzing the biosynthesis of Wax Esters and triacylglycerols. The unspecificity of WS/DGAT was used for in vitro and in vivo biosynthesis of Wax diEsters consisting of 1,16-hexadecanediol Esterified with long-chain fatty acids. An in vitro assay employing the membrane fraction of recombinant Escherichia coli XL1 -Blue expressing Wax/dgat coding for WS/DGAT and using 1,16-hexadecanediol and 1- 14 C-palmitoyl-CoA as substrates resulted in the production of 2 radiolabeled substances as revealed by autoradiography suggesting the acylation of one or both hydroxy groups of 1,16-hexadecanediol by WS/DGAT. For in vivo biosynthesis of Wax diEsters, the knock-out mutant A. calcoaceticus ADP1 acr1| Km was generated by disruption of acr1 coding for acyl-CoA reductase which caused the inability to synthesize fatty alcohols and, thus in consequence, Wax Esters. Co-cultivation of A. calcoaceticus ADP1 acr1| Km on gluconate and 1,16-hexadecanediol in nitrogen-limited mineral salts medium resulted in the accumulation of a mixture of Wax diEsters of 1,16-hexadecanediol Esterified with palmitic and oleic acid as revealed by electron impact ionization mass spectrometry. 1-Monopalmitoylglycerol could also be utilized as an alternative acyl acceptor by the unspecific WS/DGAT in vitro resulting in the synthesis of 1,2- and 1,3-dipalmitoylglycerol, whereas 1-oleoylglycerol-3-phosphate (lysophosphatidic acid) was not accepted.
Brett M Barney - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of two thioEsterases from Marinobacter aquaeolei VT8: Relationship to Wax Ester production.
FEMS microbiology letters, 2020Co-Authors: Amelia M. Lijewski, Eric M Lenneman, Carolann M. Knutson, Brett M BarneyAbstract:The biosynthesis of lipid-based biofuels is an important aspect of developing sustainable alternatives to conventional oils derived from fossil fuel reserves. Many biosynthetic approaches to biodiesel fuels and oils involve fatty acid derivatives as a precursor, and thioEsterases have been employed in various strategies to increase fatty acid pools. ThioEsterases liberate fatty acids from fatty acyl-coenzyme A or fatty acyl-acyl carrier protein substrates. The role played by thioEsterases has not been extensively studied in model bacteria that accumulate elevated levels of biological oils based on fatty acid precursors. In this report, two primary thioEsterases from the Wax Ester accumulating bacterium Marinobacter aquaeolei VT8 were heterologously expressed, isolated and characterized. These genes were further analyzed at the transcriptional level in the native bacterium during Wax Ester accumulation, and their genes were disrupted to determine the effect these changes had on Wax Ester levels. Combined, these results indicate that these two thioEsterases do not play an integral role in Wax Ester accumulation in this natural lipid-accumulating model bacterium.
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Altering small and medium alcohol selectivity in the Wax Ester synthase
Applied Microbiology and Biotechnology, 2015Co-Authors: Brett M Barney, Janet M Ohlert, Jacobe G. Timler, Amelia M. LijewskiAbstract:The bifunctional Wax Ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT or Wax Ester synthase) catalyzes the terminal reaction in the bacterial Wax Ester biosynthetic pathway, utilizing a range of alcohols and fatty acyl-CoAs to synthesize the corresponding Wax Ester. The wild-type Wax Ester synthase Maqu_0168 from Marinobacter aquaeolei VT8 exhibits a preference for longer fatty alcohols, while applications with smaller alcohols would yield products with desired biotechnological properties. Small and medium chain length alcohol substrates are much poorer substrates for the native enzyme, which may hinder broad application of the Wax Ester synthase in many proposed biosynthetic schemes. Developing approaches to improve enzyme activity toward specific smaller alcohol substrates first requires a clear understanding of which amino acids of the primary sequences of these enzymes contribute to substrate specificity in the native enzyme. In this report, we surveyed a range of potential residues and identified the leucine at position 356 and methionine at position 405 in Maqu_0168 as residues that affected selectivity toward small, branched, and aromatic alcohols when substituted with different amino acids. This analysis provides evidence of residues that line the binding site for Wax Ester synthase, which will aid rational approaches to improve this enzyme with specific substrates.
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fatty alcohols for Wax Esters in marinobacter aquaeolei vt8 two optional routes in the Wax biosynthesis pathway
Applied and Environmental Microbiology, 2013Co-Authors: Eric M Lenneman, Janet M Ohlert, Nagendra P Palani, Brett M BarneyAbstract:The biosynthesis of Wax Esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previous in vitro enzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol in Marinobacter aquaeolei VT8. 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 biosynthesis in 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 previous in vitro studies and provide the first glimpse into the role of each enzyme in vivo in the native organism.
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Identification of a Residue Affecting Fatty Alcohol Selectivity in Wax Ester Synthase
Applied and Environmental Microbiology, 2012Co-Authors: Brett M Barney, Rachel Mann, Janet M OhlertAbstract:ABSTRACT The terminal enzyme in the bacterial Wax Ester biosynthetic pathway is the bifunctional Wax Ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT), which utilizes a fatty alcohol and a fatty acyl-coenzyme A (CoA) to synthesize the corresponding Wax Ester. In this report, we identify a specific residue in WS/DGAT enzymes obtained from Marinobacter aquaeolei VT8 and Acinetobacter baylyi that alters fatty alcohol selectivity and kinetic parameters when modified to alternative residues.
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Differences in Substrate Specificities of Five Bacterial Wax Ester Synthases
Applied and Environmental Microbiology, 2012Co-Authors: Brett M Barney, Bradley D. Wahlen, Emma Lee Garner, Lance C. SeefeldtAbstract:Wax Esters are produced in certain bacteria as a potential carbon and energy storage compound. The final enzyme in the biosynthetic pathway responsible for Wax Ester production is the bifunctional Wax Ester synthase/acyl-coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), which utilizes a range of fatty alcohols and fatty acyl-CoAs to synthesize the corresponding Wax Ester. We report here the isolation and substrate range characterization for five WS/DGAT enzymes from four different bacteria: Marinobacter aquaeolei VT8, Acinetobacter baylyi, Rhodococcus jostii RHA1, and Psychrobacter cryohalolentis K5. The results from kinetic studies of isolated enzymes reveal a differential activity based on the order of substrate addition and reveal subtle differences between the substrate selectivity of the different enzymes. These in vitro results are compared to the Wax Ester and triacylglyceride product profiles obtained from each organism grown under neutral lipid accumulating conditions, providing potential insights into the role that the WS/DGAT enzyme plays in determining the final Wax Ester products that are produced under conditions of nutrient stress in each of these bacteria. Further, the analysis revealed that one enzyme in particular from M. aquaeolei VT8 showed the greatest potential for future study based on rapid purification and significantly higher activity than was found for the other isolated WS/DGAT enzymes. The results provide a framework to test prospective differences between these enzymes for potential biotechnological applications such as high-value petrochemicals and biofuel production.
Ville Santala - One of the best experts on this subject based on the ideXlab platform.
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Wax Ester production in nitrogen rich conditions by metabolically engineered acinetobacter baylyi adp1
Metabolic Engineering Communications, 2020Co-Authors: Jin Luo, Ville Santala, Elena Efimova, Pauli Losoi, Suvi SantalaAbstract:Abstract Metabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal for the production. An example of synthesis strongly dependent on external conditions is the production of storage lipids, which typically requires a high carbon/nitrogen ratio. This requirement also limits the use of abundant nitrogen-rich materials, such as industrial protein by-products, as substrates for lipid production. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely Wax Esters (WEs). Here, we engineered A. baylyi ADP1 by deleting the gene aceA encoding for isocitrate lyase and overexpressing fatty acyl-CoA reductase Acr1 in the Wax Ester production pathway to allow redirection of carbon towards WEs. This strategy led to 3-fold improvement in yield (0.075 g/g glucose) and 3.15-fold improvement in titer (1.82 g/L) and productivity (0.038 g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of Wax Esters using nitrogen-rich substrates including casamino acids, yeast extract, and baker’s yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids.
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dynamic decoupling of biomass and Wax Ester biosynthesis in acinetobacter baylyi by an autonomously regulated switch
Metabolic Engineering Communications, 2018Co-Authors: Suvi Santala, Elena Efimova, Ville SantalaAbstract:For improving the microbial production of fuels and chemicals, gene knock-outs and overexpression are routinely applied to intensify the carbon flow from substrate to product. However, their possibilities in dynamic control of the flux between the biomass and product synthesis are limited, whereas dynamic metabolic switches can be used for optimizing the distribution of carbon and resources. The production of single cell oils is especially challenging, as the synthesis is strictly regulated, competes directly with biomass, and requires defined conditions, such as nitrogen limitation. Here, we engineered a metabolic switch for redirecting carbon flow from biomass to Wax Ester production in Acinetobacter baylyi ADP1 using acetate as a carbon source. Isocitrate lyase, an essential enzyme for growth on acetate, was expressed under an arabinose inducible promoter. The autonomous downregulation of the expression is based on the gradual oxidation of the arabinose inducer by a glucose dehydrogenase gcd. The depletion of the inducer, occurring simultaneously to acetate consumption, switches the cells from a biomass mode to a lipid synthesis mode, enabling the efficient channelling of carbon to Wax Esters in a simple batch culture. In the engineered strain, the yield and titer of Wax Esters were improved by 3.8 and 3.1 folds, respectively, over the control strain. In addition, the engineered strain accumulated Wax Esters 19% of cell dry weight, being the highest reported among microbes. The study provides important insights into the dynamic engineering of the biomass-dependent synthesis pathways for the improved production of biocompounds from low-cost and sustainable substrates.
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Improved fatty aldehyde and Wax Ester production by overexpression of fatty acyl-CoA reductases
Microbial Cell Factories, 2018Co-Authors: Tapio Lehtinen, Suvi Santala, Elena Efimova, Ville SantalaAbstract:Fatty aldehydes are industrially relevant compounds, which also represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and Wax Esters. The key enzymes in biological fatty aldehyde production are the fatty acyl-CoA/ACP reductases (FARs) which reduce the activated acyl molecules to fatty aldehydes. Due to the disparity of FARs, identification and in vivo characterization of reductases with different properties are needed for the construction of tailored synthetic pathways for the production of various compounds. Fatty aldehyde production in Acinetobacter baylyi ADP1 was increased by the overexpression of three different FARs: a native A. baylyi FAR Acr1, a cyanobacterial Aar, and a putative, previously uncharacterized dehydrogenase (Ramo) from Nevskia ramosa. The fatty aldehyde production was followed in real-time inside the cells with a luminescence-based tool, and the highest aldehyde production was achieved with Aar. The fate of the overproduced fatty aldehydes was studied by measuring the production of Wax Esters by a native downstream pathway of A. baylyi, for which fatty aldehyde is a specific intermediate. The Wax Ester production was improved with the overexpression of Acr1 or Ramo compared to the wild type A. baylyi by more than two-fold, whereas the expression of Aar led to only subtle Wax Ester production. The overexpression of FARs did not affect the length of the acyl chains of the Wax Esters. The fatty aldehyde production, as well as the Wax Ester production of A. baylyi, was improved with the overexpression of a key enzyme in the pathway. The Wax Ester titer (0.45 g/l) achieved with the overexpression of Acr1 is the highest reported without hydrocarbon supplementation to the culture. The contrasting behavior of the different reductases highlight the significance of in vivo characterization of enzymes and emphasizes the possibilities provided by the diversity of FARs for pathway and product modulation.
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Improved fatty aldehyde and Wax Ester production by overexpression of fatty acyl-CoA reductases
'Springer Science and Business Media LLC', 2018Co-Authors: Tapio Lehtinen, Suvi Santala, Elena Efimova, Ville SantalaAbstract:Abstract Background Fatty aldehydes are industrially relevant compounds, which also represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and Wax Esters. The key enzymes in biological fatty aldehyde production are the fatty acyl-CoA/ACP reductases (FARs) which reduce the activated acyl molecules to fatty aldehydes. Due to the disparity of FARs, identification and in vivo characterization of reductases with different properties are needed for the construction of tailored synthetic pathways for the production of various compounds. Results Fatty aldehyde production in Acinetobacter baylyi ADP1 was increased by the overexpression of three different FARs: a native A. baylyi FAR Acr1, a cyanobacterial Aar, and a putative, previously uncharacterized dehydrogenase (Ramo) from Nevskia ramosa. The fatty aldehyde production was followed in real-time inside the cells with a luminescence-based tool, and the highest aldehyde production was achieved with Aar. The fate of the overproduced fatty aldehydes was studied by measuring the production of Wax Esters by a native downstream pathway of A. baylyi, for which fatty aldehyde is a specific intermediate. The Wax Ester production was improved with the overexpression of Acr1 or Ramo compared to the wild type A. baylyi by more than two-fold, whereas the expression of Aar led to only subtle Wax Ester production. The overexpression of FARs did not affect the length of the acyl chains of the Wax Esters. Conclusions The fatty aldehyde production, as well as the Wax Ester production of A. baylyi, was improved with the overexpression of a key enzyme in the pathway. The Wax Ester titer (0.45 g/l) achieved with the overexpression of Acr1 is the highest reported without hydrocarbon supplementation to the culture. The contrasting behavior of the different reductases highlight the significance of in vivo characterization of enzymes and emphasizes the possibilities provided by the diversity of FARs for pathway and product modulation
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Dynamic decoupling of biomass and Wax Ester biosynthesis in Acinetobacter baylyi by an autonomously regulated switch
Elsevier, 2018Co-Authors: Suvi Santala, Elena Efimova, Ville SantalaAbstract:For improving the microbial production of fuels and chemicals, gene knock-outs and overexpression are routinely applied to intensify the carbon flow from substrate to product. However, their possibilities in dynamic control of the flux between the biomass and product synthesis are limited, whereas dynamic metabolic switches can be used for optimizing the distribution of carbon and resources. The production of single cell oils is especially challenging, as the synthesis is strictly regulated, competes directly with biomass, and requires defined conditions, such as nitrogen limitation. Here, we engineered a metabolic switch for redirecting carbon flow from biomass to Wax Ester production in Acinetobacter baylyi ADP1 using acetate as a carbon source. Isocitrate lyase, an essential enzyme for growth on acetate, was expressed under an arabinose inducible promoter. The autonomous downregulation of the expression is based on the gradual oxidation of the arabinose inducer by a glucose dehydrogenase gcd. The depletion of the inducer, occurring simultaneously to acetate consumption, switches the cells from a biomass mode to a lipid synthesis mode, enabling the efficient channelling of carbon to Wax Esters in a simple batch culture. In the engineered strain, the yield and titer of Wax Esters were improved by 3.8 and 3.1 folds, respectively, over the control strain. In addition, the engineered strain accumulated Wax Esters 19% of cell dry weight, being the highest reported among microbes. The study provides important insights into the dynamic engineering of the biomass-dependent synthesis pathways for the improved production of biocompounds from low-cost and sustainable substrates. Keywords: Lipid biosynthesis, Wax Esters, Acetate, Dynamic control, Decoupling, Autonomous circui
Rainer Kalscheuer - One of the best experts on this subject based on the ideXlab platform.
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both histidine residues of the conserved hhxxxdg motif are essential for Wax Ester synthase acyl coa diacylglycerol acyltransferase catalysis
European Journal of Lipid Science and Technology, 2009Co-Authors: Tim Stoveken, Rainer Kalscheuer, Alexander SteinbuchelAbstract:Bacterial acyltransferases of the Wax Ester synthase/diacylglycerol acyltransferase (WS/DGAT) family possess a highly conserved HHXXXDG motif. In this study, we describe the first experimental evidence that this motif is part of the active site of WS/DGAT from the Acinetobacter baylyi strain ADP1 and that it is crucial for enzymatic activity. The second histidine residue of this motif (H133) turned out to be essential for the catalytic activity. In addition, the replacement of the first histidine (His132) also led to explicitly decreased activity. A complete loss of activity was only observed upon substitution of both histidine residues by leucine, revealing that both are necessary for maximal activity. In contrast, the replacement of Asp137 and Gly138 against alanine had only little effect on enzyme activity, thus demonstrating that they are not essential for WS/DGAT catalysis although belonging to the highly conserved motif. One peculiarity of WS/DGAT enzymes is their little substrate specificity regarding hydrophobic compounds. In this study, we demonstrated the inability of WS/DGAT to accept polar compounds as substrates.
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the Wax Ester synthase acyl coenzyme a diacylglycerol acyltransferase from acinetobacter sp strain adp1 characterization of a novel type of acyltransferase
Journal of Bacteriology, 2005Co-Authors: Tim Stoveken, Rainer Kalscheuer, Ursula Malkus, Rudolf Reichelt, Alexander SteinbuchelAbstract: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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thio Wax Ester biosynthesis utilizing the unspecific bifunctional Wax Ester synthase acyl coenzyme a diacylglycerol acyltransferase of acinetobacter sp strain adp1
Applied and Environmental Microbiology, 2005Co-Authors: Stefan Uthoff, Tim Stoveken, N Weber, Klaus Vosmann, Erika Klein, Rainer Kalscheuer, Alexander SteinbuchelAbstract: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′His6atf). 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.
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in vitro and in vivo biosynthesis of Wax diEsters by an unspecific bifunctional Wax Ester synthase acyl coa diacylglycerol acyltransferase from acinetobacter calcoaceticus adp1
European Journal of Lipid Science and Technology, 2003Co-Authors: Rainer Kalscheuer, Stefan Uthoff, Heinrich Luftmann, Alexander SteinbuchelAbstract:Acinetobacter calcoaceticus ADP1 possesses a bifunctional Wax Ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) catalyzing the biosynthesis of Wax Esters and triacylglycerols. The unspecificity of WS/DGAT was used for in vitro and in vivo biosynthesis of Wax diEsters consisting of 1,16-hexadecanediol Esterified with long-chain fatty acids. An in vitro assay employing the membrane fraction of recombinant Escherichia coli XL1 -Blue expressing Wax/dgat coding for WS/DGAT and using 1,16-hexadecanediol and 1- 14 C-palmitoyl-CoA as substrates resulted in the production of 2 radiolabeled substances as revealed by autoradiography suggesting the acylation of one or both hydroxy groups of 1,16-hexadecanediol by WS/DGAT. For in vivo biosynthesis of Wax diEsters, the knock-out mutant A. calcoaceticus ADP1 acr1| Km was generated by disruption of acr1 coding for acyl-CoA reductase which caused the inability to synthesize fatty alcohols and, thus in consequence, Wax Esters. Co-cultivation of A. calcoaceticus ADP1 acr1| Km on gluconate and 1,16-hexadecanediol in nitrogen-limited mineral salts medium resulted in the accumulation of a mixture of Wax diEsters of 1,16-hexadecanediol Esterified with palmitic and oleic acid as revealed by electron impact ionization mass spectrometry. 1-Monopalmitoylglycerol could also be utilized as an alternative acyl acceptor by the unspecific WS/DGAT in vitro resulting in the synthesis of 1,2- and 1,3-dipalmitoylglycerol, whereas 1-oleoylglycerol-3-phosphate (lysophosphatidic acid) was not accepted.
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a novel bifunctional Wax Ester synthase acyl coa diacylglycerol acyltransferase mediates Wax Ester and triacylglycerol biosynthesis in acinetobacter calcoaceticus adp1
Journal of Biological Chemistry, 2003Co-Authors: Rainer Kalscheuer, Alexander SteinbuchelAbstract:Abstract Triacylglycerols (TAGs) and Wax Esters are neutral lipids with considerable importance for dietetic, technical, cosmetic, and pharmaceutical applications. Acinetobacter calcoaceticus ADP1 accumulates Wax Esters and TAGs as intracellular storage lipids. We describe here the identification of a bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty alcohol acyltransferase (Wax Ester synthase, WS) as well as acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. Experiments with a knock-out mutant demonstrated the key role of the bifunctional WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA acyltransferase is not related to known acyltransferases including the WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2 families present in yeast, plants, and animals, and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in yeast and plants. A large number of WS/DGAT-related proteins were identified in Mycobacterium and Arabidopsis thaliana indicating an important function of these proteins. WS and DGAT activity was demonstrated for the translational product of one WS/DGAT homologous gene from M. smegmatismc2155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like Wax Esters was demonstrated by heterologous expression in recombinantPseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed.
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high level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient Wax Ester synthesis enzymes
Biotechnology for Biofuels, 2018Co-Authors: Ellen Hornung, Tim Iven, Ivo FeussnerAbstract:Biotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived Wax Esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of Wax Esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a Wax synthase (WS) in seeds are sufficient. For optimization of the enzymatic activity and subcellular localization of Wax Ester synthesis enzymes, two fusion proteins were created, which showed Wax Ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for Wax Ester production. The tested seven enzyme combinations resulted in different yields and compositions of Wax Esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in Wax Esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into Wax Esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total Wax Esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed−1 of Wax Esters, containing 27–34 mol% oleyl oleate. The overall yields and the compositions of Wax Esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of Wax Ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate Wax Ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds.
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High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient Wax Ester synthesis enzymes
BMC, 2018Co-Authors: Ellen Hornung, Tim Iven, Ivo FeussnerAbstract:Abstract Background Biotechnology enables the production of high-valued industrial feedstocks from plant seed oil. The plant-derived Wax Esters with long-chain monounsaturated acyl moieties, like oleyl oleate, have favorite properties for lubrication. For biosynthesis of Wax Esters using acyl-CoA substrates, expressions of a fatty acyl reductase (FAR) and a Wax synthase (WS) in seeds are sufficient. Results For optimization of the enzymatic activity and subcellular localization of Wax Ester synthesis enzymes, two fusion proteins were created, which showed Wax Ester-forming activities in Saccharomyces cerevisiae. To promote the formation of oleyl oleate in seed oil, WSs from Acinetobactor baylyi (AbWSD1) and Marinobacter aquaeolei (MaWS2), as well as the two created fusion proteins were tested in Arabidopsis to evaluate their abilities and substrate preference for Wax Ester production. The tested seven enzyme combinations resulted in different yields and compositions of Wax Esters. Expression of a FAR of Marinobacter aquaeolei (MaFAR) with AbWSD1 or MaWS2 led to a high incorporation of C18 substrates in Wax Esters. The MaFAR/TMMmAWAT2-AbWSD1 combination resulted in the incorporation of more C18:1 alcohol and C18:0 acyl moieties into Wax Esters compared with MaFAR/AbWSD1. The fusion protein of a WS from Simmondsia chinensis (ScWS) with MaFAR exhibited higher specificity toward C20:1 substrates in preference to C18:1 substrates. Expression of MaFAR/AbWSD1 in the Arabidopsis fad2 fae1 double mutant resulted in the accumulation of oleyl oleate (18:1/18:1) in up to 62 mol% of total Wax Esters in seed oil, which was much higher than the 15 mol% reached by MaFAR/AbWSD1 in Arabidopsis Col-0 background. In order to increase the level of oleyl oleate in seed oil of Camelina, lines expressing MaFAR/ScWS were crossed with a transgenic high oleate line. The resulting plants accumulated up to >40 mg g seed−1 of Wax Esters, containing 27–34 mol% oleyl oleate. Conclusions The overall yields and the compositions of Wax Esters can be strongly affected by the availability of acyl-CoA substrates and to a lesser extent, by the characteristics of Wax Ester synthesis enzymes. For synthesis of oleyl oleate in plant seed oil, appropriate Wax Ester synthesis enzymes with high catalytic efficiency and desired substrate specificity should be expressed in plant cells; meanwhile, high levels of oleic acid-derived substrates need to be supplied to these enzymes by modifying the fatty acid profile of developing seeds
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MOESM10 of High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient Wax Ester synthesis enzymes
2018Co-Authors: Ellen Hornung, Tim Iven, Ivo FeussnerAbstract:Additional file 10: Table S6. List of DNA constructs used in this study for expressing Wax Ester synthesis enzymes
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MOESM2 of High-level accumulation of oleyl oleate in plant seed oil by abundant supply of oleic acid substrates to efficient Wax Ester synthesis enzymes
2018Co-Authors: Ellen Hornung, Tim Iven, Ivo FeussnerAbstract:Additional file 2: Table S1. Raw data obtained by GC-FID to quantify the Wax Ester content in seeds of transgenic Arabidopsis transformed with MaFAR/ScWS, ScWS-MaFAR, ScWS-MaFAR/ScWS-MaFAR, ScWS-MaFAR/MaFAR, MaFAR/AbWSD1, MaFAR/PCOAbWSD1, MaFAR/TMMmAWAT2-AbWSD1 and MaFAR/MaWS2 shown in Figs. 1 and 3
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production of Wax Esters in the wild oil species lepidium campestre
Industrial Crops and Products, 2017Co-Authors: Emelie Ivarson, Annelie Ahlman, Kent D Chapman, Drew Sturtevant, Tim Iven, Ivo FeussnerAbstract:Abstract The objective of this study was to evaluate whether Wax Esters could be produced, and to determine what types of Wax Esters could be produced in the wild oil species field cress ( Lepidium campestre L.). Wax Esters produced in plants are perhaps best suited for lubrication applications; however, natural sources of plant-derived Wax Esters are limited and new resources need to be generated. Moreover, the composition of Wax Esters needs to be optimized in planta in order to suit various end uses for commercial applications. In this study, three jojoba Wax Ester synthesis genes under a seed specific promoter were introduced into field cress. The results show that expressing the jojoba genes produced various amounts of Wax Esters in the seed oil of field cress with C42 and C44 as the most abundant Wax Ester molecular species. The additional expression of a jojoba fatty acyl-coenzyme A elongase resulted in an increased level of longer chain C46 and C48 Wax Esters. The cellular distribution of neutral lipids appeared to be disrupted in the transgenic seeds with high Wax Ester content compared with wild type and Wax Esters were mainly localized in the seed coat. Collectively, the results indicate that field cress has the potential to be developed as a new industrial oil crop for Wax Ester production, and an improved understanding of mechanisms for the correct packaging of Wax Esters in seeds may influence Wax Ester amounts accumulated in transgenic crops.
