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 Use of Limited Proteolysis and Mutagenesis To Identify Folding Domains and Sequence Motifs Critical for Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase Activity

2013 ◽  
Vol 80 (3) ◽  
pp. 1132-1141 ◽  
Author(s):  
Juan A. Villa ◽  
Matilde Cabezas ◽  
Fernando de la Cruz ◽  
Gabriel Moncalián
Keyword(s):  
Neutral Lipids ◽  
Limited Proteolysis ◽  
Diacylglycerol Acyltransferase ◽  
Wax Ester ◽  
Sequence Motifs ◽  
Acyltransferase Activity ◽  
Structural Prediction ◽  
Active Complex ◽  
Acyl Coenzyme A ◽  
Wax Ester Synthase

ABSTRACTTriacylglycerols and wax esters are synthesized as energy storage molecules by some proteobacteria and actinobacteria under stress. The enzyme responsible for neutral lipid accumulation is the bifunctional wax ester synthase/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT). Structural modeling of WS/DGAT suggests that it can adopt an acyl-CoA-dependent acyltransferase fold with the N-terminal and C-terminal domains connected by a helical linker, an architecture demonstrated experimentally by limited proteolysis. Moreover, we found that both domains form an active complex when coexpressed as independent polypeptides. The structural prediction and sequence alignment of different WS/DGAT proteins indicated catalytically important motifs in the enzyme. Their role was probed by measuring the activities of a series of alanine scanning mutants. Our study underscores the structural understanding of this protein family and paves the way for their modification to improve the production of neutral lipids.

scholarly journals Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

2010 ◽  
Vol 76 (9) ◽  
pp. 2884-2894 ◽  
Author(s):  
Efraín Manilla-Pérez ◽  
Alvin Brian Lange ◽  
Stephan Hetzler ◽  
Marc Wältermann ◽  
Rainer Kalscheuer ◽  
...  
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Marine Bacterium ◽  
Neutral Lipids ◽  
Nile Red ◽  
Dry Weight ◽  
Wax Ester ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Key Enzyme

ABSTRACT In many microorganisms, the key enzyme responsible for catalyzing the last step in triacylglycerol (TAG) and wax ester (WE) biosynthesis is an unspecific acyltransferase which is also referred to as wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT; AtfA). The importance and function of two AtfA homologues (AtfA1 and AtfA2) in the biosynthesis of TAGs and WEs in the hydrocarbon-degrading marine bacterium Alcanivorax borkumensis SK2 have been described recently. However, after the disruption of both the AtfA1 and AtfA2 genes, reduced but substantial accumulation of TAGs was still observed, indicating the existence of an alternative TAG biosynthesis pathway. In this study, transposon-induced mutagenesis was applied to an atfA1 atfA2 double mutant to screen for A. borkumensis mutants totally defective in biosynthesis of neutral lipids in order to identify additional enzymes involved in the biosynthesis of these lipids. At the same time, we have searched for a totally TAG-negative mutant in order to study the function of TAGs in A. borkumensis. Thirteen fluorescence-negative mutants were identified on Nile red ONR7a agar plates and analyzed for their abilities to synthesize lipids. Among these, mutant 2 M131 was no longer able to synthesize and accumulate TAGs if pyruvate was used as the sole carbon source. The transposon insertion was localized in a gene encoding a putative cytochrome c family protein (ABO_1185). Growth and TAG accumulation experiments showed that the disruption of this gene resulted in the absence of TAGs in 2 M131 but that growth was not affected. In cells of A. borkumensis SK2 grown on pyruvate as the sole carbon source, TAGs represented about 11% of the dry weight of the cells, while in the mutant 2 M131, TAGs were not detected by thin-layer and gas chromatography analyses. Starvation and lipid mobilization experiments revealed that the lipids play an important role in the survival of the cells. The function of neutral lipids in A. borkumensis SK2 is discussed.

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.

scholarly journals Mutations in the midway Gene Disrupt a Drosophila Acyl Coenzyme A: Diacylglycerol Acyltransferase

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1511-1518
Author(s):  
Michael Buszczak ◽  
Xiaohui Lu ◽  
William A Segraves ◽  
Ta Yuan Chang ◽  
Lynn Cooley
Keyword(s):  
Cell Death ◽  
Insect Cells ◽  
Coenzyme A ◽  
Neutral Lipids ◽  
Diacylglycerol Acyltransferase ◽  
Nurse Cells ◽  
Germline Expression ◽  
Egg Chambers ◽  
Acyl Coenzyme A

Abstract During Drosophila oogenesis, defective or unwanted egg chambers are eliminated during mid-oogenesis by programmed cell death. In addition, final cytoplasm transport from nurse cells to the oocyte depends upon apoptosis of the nurse cells. To study the regulation of germline apoptosis, we analyzed the midway mutant, in which egg chambers undergo premature nurse cell death and degeneration. The midway gene encodes a protein similar to mammalian acyl coenzyme A: diacylglycerol acyltransferase (DGAT), which converts diacylglycerol (DAG) into triacylglycerol (TAG). midway mutant egg chambers contain severely reduced levels of neutral lipids in the germline. Expression of midway in insect cells results in high levels of DGAT activity in vitro. These results show that midway encodes a functional DGAT and that changes in acylglycerol lipid metabolism disrupt normal egg chamber development in Drosophila.

scholarly journals Identification of a Residue Affecting Fatty Alcohol Selectivity in Wax Ester Synthase

2012 ◽  
Vol 79 (1) ◽  
pp. 396-399 ◽  
Author(s):  
Brett M. Barney ◽  
Rachel L. Mann ◽  
Janet M. Ohlert
Keyword(s):  
Kinetic Parameters ◽  
Fatty Alcohol ◽  
Biosynthetic Pathway ◽  
Fatty Acyl ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Content Type ◽  
Marinobacter Aquaeolei ◽  
Acyl Coenzyme A ◽  
Wax Ester Synthase

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.

scholarly journals Biosynthesis of Long Chain Alkyl Diols and Long Chain Alkenols in Nannochloropsis spp. (Eustigmatophyceae)

2019 ◽  
Vol 60 (8) ◽  
pp. 1666-1682
Author(s):  
Sergio Balzano ◽  
Laura Villanueva ◽  
Marijke de Bar ◽  
Diana X Sahonero Canavesi ◽  
Caglar Yildiz ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Monounsaturated Fatty Acids ◽  
Wax Ester ◽  
Biosynthetic Pathways ◽  
Nannochloropsis Gaditana ◽  
Acyl Coenzyme A ◽  
Wax Ester Synthase ◽  
Long Chain

AbstractWe investigated potential biosynthetic pathways of long chain alkenols (LCAs), long chain alkyl diols (LCDs), and long chain hydroxy fatty acids (LCHFAs) in Nannochloropsis oceanica and Nannochloropsis gaditana, by combining culturing experiments with genomic and transcriptomic analyses. Incubation of Nannochloropsis spp. in the dark for 1 week led to significant increases in the cellular concentrations of LCAs and LCDs in both species. Consistently, 13C-labelled substrate experiments confirmed that both LCA and LCD were actively produced in the dark from C14–18 fatty acids by either condensation or elongation/hydroxylation, although no enzymatic evidence was found for the former pathway. Nannochloropsis spp. did, however, contain (i) multiple polyketide synthases (PKSs) including one type (PKS-Clade II) that might catalyze incomplete fatty acid elongations leading to the formation of 3-OH-fatty acids, (ii) 3-hydroxyacyl dehydratases (HADs), which can possibly form Δ2/Δ3 monounsaturated fatty acids, and (iii) fatty acid elongases (FAEs) that could elongate 3-OH-fatty acids and Δ2/Δ3 monounsaturated fatty acids to longer products. The enzymes responsible for reduction of the long chain fatty acids to LCDs and LCAs are, however, unclear. A putative wax ester synthase/acyl coenzyme A (acyl-CoA): diacylglycerol acyltransferase is likely to be involved in the esterification of LCAs and LCDs in the cell wall. Our data thus provide useful insights in predicting the biosynthetic pathways of LCAs and LCDs in phytoplankton suggesting a key role of FAE and PKS enzymes.

scholarly journals Nile Red Incubation Time Before Reading Fluorescence Greatly Influences the Yeast Neutral Lipids Quantification

2021 ◽  
Vol 12 ◽  
Author(s):  
Mauricio Ramírez-Castrillón ◽  
Victoria P. Jaramillo-Garcia ◽  
Helio Lopes Barros ◽  
João A. Pegas Henriques ◽  
Valter Stefani ◽  
...  
Keyword(s):  
Lipid Content ◽  
High Throughput Screening ◽  
Oleaginous Yeast ◽  
Neutral Lipids ◽  
Nile Red ◽  
Oleaginous Yeasts ◽  
Red Fluorescence ◽  
Fluorescence Measurements ◽  
Yeast Strains ◽  
Nile Red Fluorescence

High-throughput screening methodologies to estimate lipid content in oleaginous yeasts use Nile red fluorescence in a given solvent and optimized excitation/emission wavelengths. However, Nile red fluorescence stabilization has been poorly analyzed, and high variability occurs when relative fluorescence is measured immediately or a few minutes after dye addition. The aim of this work was to analyze the fluorescence of Nile red at different incubation times using a variety of solvents and oleaginous/non-oleaginous yeast strains. We showed that fluorescence stabilization occurs between 20 and 30 min, depending on the strain and solvent. Therefore, we suggest that fluorescence measurements should be followed until stabilization, where Relative Fluorescence Units should be considered after stabilization for lipid content estimation.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

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