In vitro characterization of five bacterial WS/DGAT acyltransferases regarding the synthesis of biotechnologically relevant short‐chain‐length esters

Röttig, Annika; Wolf, Sebastián; Steinbüchel, Alexander

doi:10.1002/ejlt.201500200

Cited by 14 publications

(21 citation statements)

References 20 publications

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“…To identify new WSD enzymes in the bacterium M. aquaeolei VT8, a BLAST homology search on the genome of M. aquaeolei VT8 using the amino acid sequence of AbWSD1 was performed. In addition to the four already known and partly characterized WSD MaWSD1, MaWSD2, MaWSD3, and MaWSD4 (Barney et al, 2012(Barney et al, , 2013(Barney et al, , 2015Petronikolou and Nair, 2018;Röttig et al, 2016;Villa et al, 2014), a putative fifth WSD (MaWSD5, Maqu_3411) was identified. While characterizing MaWSD5, the existence of the protein was also mentioned in a book chapter (Knutson et al, 2017).…”

Section: Aquaeolei Vt8 Possesses a Fifth Wsdmentioning

confidence: 95%

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8

Vollheyde¹,

Yu²,

Hornung³

et al. 2020

Lipids

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Wax esters (WE) belong to the class of neutral lipids. They are formed by an esterification of a fatty alcohol and an activated fatty acid. Dependent on the chain length and desaturation degree of the fatty acid and the fatty alcohol moiety, WE can have diverse physicochemical properties. WE derived from monounsaturated long-chain acyl moieties are of industrial interest due to their very good lubrication properties. Whereas WE were obtained in the past from spermaceti organs of the sperm whale, industrial WE are nowadays mostly produced chemically from fossil fuels. In order to produce WE more sustainably, attempts to produce industrial WE in transgenic plants are steadily increasing. To achieve this, different combinations of WE producing enzymes are expressed in developing Arabidopsis thaliana or Camelina sativa seeds. Here we report the identification and characterization of a fifth wax synthase from the organism Marinobacter aquaeolei VT8, MaWSD5. It belongs to the class of bifunctional wax synthase/acyl-CoA:diacylglycerol O-acyltransferases (WSD). The protein was purified to homogeneity. In vivo and in vitro substrate analyses revealed that MaWSD5 is able to synthesize WE but no triacylglycerols. The protein produces WE from saturated and monounsaturated mid-and long-chain substrates. Arabidopsis thaliana seeds expressing a fatty acid reductase from Marinobacter aquaeolei VT8 and MaWSD5 produce WE. Main WE synthesized are 20:1/18:1 and 20:1/ 20:1. This makes MaWSD5 a suitable candidate for industrial WE production in planta. Keywords Marinobacter aquaeolei VT8 Á Neutral lipids Á Seed oil Á Storage lipids Á Wax ester Á Wax synthase/acyl CoA:diacylglycerol O-acyltransferase Lipids (2020) 55: 479-494. Abbreviations A. baylyi Acinetobacter baylyi A. calcoaceticus Acinetobacter calcoaceticus, nowadays: Acinetobacter baylyi A. thaliana Arabidopsis thaliana aa amino acids AbWSD1 Acinetobacter baylyi WSD1, Ac1, AtfA ACP acyl carrier protein ATP adenosine triphosphate C. sativa Camelina sativa Supporting information Additional supporting information may be found online in the Supporting Information section at the end of the article.

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Section: Aquaeolei Vt8 Possesses a Fifth Wsdmentioning

confidence: 95%

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8

Vollheyde¹,

Yu²,

Hornung³

et al. 2020

Lipids

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“…Atf1 and AtfA can accept a wide range of alcohols, whereas Eeb1 seems restricted to shortchain alcohols (33)(34)(35).…”

Section: Introductionmentioning

confidence: 99%

Biocatalytic, one-pot diterminal oxidation and esterification of n-alkanes for production of α,ω-diol and α,ω-dicarboxylic acid esters

Nuland

Vogel

Scott

et al. 2017

Metabolic Engineering

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Direct and selective terminal oxidation of medium-chain n-alkanes is a major challenge in chemistry. Efforts to achieve this have so far resulted in low specificity and overoxidized products. Biocatalytic oxidation of medium-chain n-alkanes - with for example the alkane monooxygenase AlkB from P. putida GPo1- on the other hand is highly selective. However, it also results in overoxidation. Moreover, diterminal oxidation of medium-chain n-alkanes is inefficient. Hence, α,ω-bifunctional monomers are mostly produced from olefins using energy intensive, multi-step processes. By combining biocatalytic oxidation with esterification we drastically increased diterminal oxidation upto 92mol% and reduced overoxidation to 3% for n-hexane. This methodology allowed us to convert medium-chain n-alkanes into α,ω-diacetoxyalkanes and esterified α,ω-dicarboxylic acids. We achieved this in a one-pot reaction with resting-cell suspensions of genetically engineered Escherichia coli. The combination of terminal oxidation and esterification constitutes a versatile toolbox to produce α,ω-bifunctional monomers from n-alkanes.

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“…The specific enzyme activity of purified acyltransferase was measured indirectly using 5,5=-dithiobis-(2-nitrobenzoic acid) (DTNB) at 37°C (38). The cuvette was filled with 720 l prewarmed buffer (50 mM sodium phosphate, pH 7.4), 100 l of a 2 mM DTNB solution in buffer, 120 l dimethyl sulfoxide (DMSO), 20 l of the acyl-CoA stock solution (1 mM in DMSO), and 20 l of a linear alcohol (4 mM in DMSO) or 1,2-dipalmitoyl-sn-glycerol (DPG; 5 mM in DMSO).…”

Section: Methodsmentioning

confidence: 99%

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

Röttig

Strittmatter

Schauer

et al. 2016

Appl Environ Microbiol

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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 Atf G25 showed acyltransferase activity with C 12 -or C 16 -acyl-CoA, C 12 to C 18 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 Atf G25 on lipid accumulation, the respective gene, atf G25 , 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 Atf G25 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. IMPORTANCEA 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. Even extreme environments, such as arid, desert-like regions, are populated by prokaryotes. These mostly Gram-positive bacteria are subjected to extreme fluctuations of temperature and water supply. In addition to desiccation and the resulting cellular damage, the bacteria frequently encounter nutrient limitation. To withstand these harsh environmental conditions, the majority of bacteria have evolved various strategies and are able to store lipophilic compounds such as polyhydroxyalkanoates (PHA), triacylglycerols (TAG), or wax esters (WE) (1-3). TAG are synthesized as primary storage compounds by many actinomycetes-which represent the pre...

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In vitro characterization of five bacterial WS/DGAT acyltransferases regarding the synthesis of biotechnologically relevant short‐chain‐length esters

Cited by 14 publications

References 20 publications

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8

The Fifth WS/DGAT Enzyme of the Bacterium Marinobacter aquaeolei VT8

Biocatalytic, one-pot diterminal oxidation and esterification of n-alkanes for production of α,ω-diol and α,ω-dicarboxylic acid esters

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

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