scholarly journals Identification of genes coding for putative wax ester synthase/diacylglycerol acyltransferase enzymes in terrestrial and marine environments

AMB Express ◽  
2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Mariana P Lanfranconi ◽  
Adrián F Alvarez ◽  
Héctor M Alvarez
Keyword(s):  
Diacylglycerol Acyltransferase ◽  
Wax Ester ◽  
Marine Environments ◽  
Wax Ester Synthase

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 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.

Altering small and medium alcohol selectivity in the wax ester synthase

2015 ◽  
Vol 99 (22) ◽  
pp. 9675-9684 ◽  
Author(s):  
Brett M. Barney ◽  
Janet M. Ohlert ◽  
Jacobe G. Timler ◽  
Amelia M. Lijewski
Keyword(s):  
Wax Ester ◽  
Wax Ester Synthase

scholarly journals Engineering oleaginous yeast Rhodotorula toruloides for overproduction of fatty acid ethyl esters

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yang Zhang ◽  
Jie Peng ◽  
Huimin Zhao ◽  
Shuobo Shi
Keyword(s):  
Fatty Acid ◽  
Oleaginous Yeast ◽  
Batch Cultivation ◽  
Fatty Acid Ethyl Esters ◽  
Ethyl Esters ◽  
Wax Ester ◽  
Fed Batch ◽  
Acinetobacter Baylyi ◽  
Wax Ester Synthase ◽  
Fed Batch Cultivation

Abstract Background Production of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70% cell mass as lipids, Rhodotorula toruloides represents an attractive host for FAEEs production. Results We first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase/acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs through shake-flask cultivation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, to elevate the performance of Δku70-AbWS* in a bioreactor, both batch and fed-batch cultivation strategies were performed. The FAEEs titer, productivity and yield were 4.03 g/L, 69.5 mg/L/h and 57.9 mg/g (mg FAEEs/g glucose) under batch cultivation, and 9.97 g/L, 90.6 mg/L/h, and 86.1 mg/g under fed-batch cultivation. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing. Conclusion We achieved the highest FAEEs production in yeast with a final titer of 9.97 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid-derived molecules.

scholarly journals Engineering Oleaginous Yeast Rhodosporidium Toruloides for Overproduction of Fatty Acid Ethyl Esters

2020 ◽  
Author(s):  
Yang Zhang ◽  
Jie Peng ◽  
Huimin Zhao ◽  
Shuobo Shi
Keyword(s):  
Fatty Acid ◽  
Oleaginous Yeast ◽  
Rhodosporidium Toruloides ◽  
Fatty Acid Ethyl Esters ◽  
Ethyl Esters ◽  
Wax Ester ◽  
Attractive Alternative ◽  
Efficient Production ◽  
Acinetobacter Baylyi ◽  
Wax Ester Synthase

Abstract BackgroundProduction of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70 % biomass as lipids, Rhodosporidium toruloides represents an attractive host for FAEEs production. ResultsWe first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase /acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs in shake-flask fermentation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, by fed-batch fermentation in a 1-L fermenter, the titer of FAEEs reached 9.2 g/L. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing.ConclusionWe achieved the highest FAEEs production in yeast with a final titer of 9.2 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid derived molecules.

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