Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana

Aslan, Selçuk; Sun, Chuanxin; Леонова, Светлана; Dutta, Paresh C.; Dörmann, Peter; Domergue, Frédéric; Stymne, Sten; Hofvander, Per

doi:10.1016/j.ymben.2014.07.001

Cited by 29 publications

(43 citation statements)

References 49 publications

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“…Using electron ionization mass spectrometry, we analyzed the WEs using the RCOOH 2 ϩ and RCO-H ϩ· ions of their saturated and unsaturated fatty acid components, respectively (29). For example, the length of the fatty acyl component of the C 32 WEs varied from 14 to 18 carbons as seen from the RCOOH 2 ϩ ions with m/z values of 229, 243, 257, 271, and 285, respectively (Fig. 2B).…”

Section: Production Of Wes In Rha1mentioning

confidence: 99%

“…Production, purification, and characterization of recombinant FcrA. We focused further efforts on exploring the function of FcrA, as related two-domain enzymes completely reduce acyl-CoAs to fatty alcohols (28) and have been used in biotechnology applications (31,32). To investigate the activity of FcrA, we produced it as a C-terminally His-tagged protein in RHA1 and purified it to greater than 95% apparent homogeneity (Fig.…”

Section: Production Of Wes In Rha1mentioning

confidence: 99%

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A Fatty Acyl Coenzyme A Reductase Promotes Wax Ester Accumulation in Rhodococcus jostii RHA1

Round

Roccor

et al. 2017

Appl Environ Microbiol

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Many rhodococci are oleaginous and, as such, have considerable potential for the sustainable production of lipid-based commodity chemicals. Herein, we demonstrated that Rhodococcus jostii RHA1, a soil bacterium that catabolizes a wide range of organic compounds, produced wax esters (WEs) up to 0.0002% of its cellular dry weight during exponential growth on glucose. These WEs were fully saturated and contained primarily 31 to 34 carbon atoms. Moreover, they were present at higher levels during exponential growth than under lipid-accumulating conditions. Bioinformatics analyses revealed that RHA1 contains a gene encoding a putative fatty acyl coenzyme A (acyl-CoA) reductase (FcrA). The purified enzyme catalyzed the NADPH-dependent transformation of stearoyl-CoA to stearyl alcohol with a specific activity of 45 Ϯ 3 nmol/mg · min and dodecanal to dodecanol with a specific activity of 5,300 Ϯ 300 nmol/mg · min. Deletion of fcrA did not affect WE accumulation when grown in either carbon-or nitrogen-limited medium. However, the ΔfcrA mutant accumulated less than 20% of the amount of WEs as the wild-type strain under conditions of nitric oxide stress. A strain of RHA1 overproducing FcrA accumulated WEs to ϳ13% cellular dry weight under lipid-accumulating conditions, and their acyl moieties had longer average chain lengths than those in wild-type cells (C 17 versus C 16 ). The results provide insight into the biosynthesis of WEs in rhodococci and facilitate the development of this genus for the production of highvalue neutral lipids. IMPORTANCE Among the best-studied oleaginous bacteria, rhodococci have considerable potential for the sustainable production of lipid-based commodity chemicals, such as wax esters. However, many aspects of lipid synthesis in these bacteria are poorly understood. The current study identifies a key enzyme in wax ester synthesis in rhodococci and exploits it to significantly improve the yield of wax esters in bacteria. In so doing, this work contributes to the development of novel bioprocesses for an important class of oleochemicals that may ultimately allow us to phase out their unsustainable production from sources such as petroleum and palm oil.KEYWORDS fatty acyl-CoA reductase, lipid accumulation, Rhodococcus, wax esters, metabolic engineering M any bacterial species, particularly the mycolic acid-producing Actinobacteria, synthesize neutral lipids for energy storage. For example, the soil bacterium Rhodococcus jostii RHA1 (referred to as RHA1 here) accumulates neutral lipids up to 70% of cellular dry weight (CDW) in response to environmental stresses, such as nitrogen limitation (1-3). These neutral lipids are primarily triacylglycerides (TAGs) and are stored in lipid bodies within the cytoplasm (4). Proteins associated with these carbon storage organelles (5) facilitate the dynamic shuffling of lipids between utilization and storage, allowing the bacterium to sequester intracellular energy reserves as needed. Due in

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Section: Production Of Wes In Rha1mentioning

confidence: 99%

Section: Production Of Wes In Rha1mentioning

confidence: 99%

A Fatty Acyl Coenzyme A Reductase Promotes Wax Ester Accumulation in Rhodococcus jostii RHA1

Round

Roccor

et al. 2017

Appl Environ Microbiol

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“…Currently, WEs have multiple important technical applications in a variety of areas such as medicine, cosmetics, and food industries, and have the potential for cost-effective and sustainable production of lubricants [12, 13]. Depending on the chain lengths and the degree of unsaturation of fatty acids and fatty alcohol components, WEs have different physical and chemical properties such as melting temperature, oxidation stability, and pressure stability [14]. Liquid WEs consisting of medium to long-chain monounsaturated fatty chains exhibit low melting-points and high oxidation stability, which renders it more suited for lubrication.…”

Section: Introductionmentioning

confidence: 99%

Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis

Zhang

Mao

Luo

et al. 2017

Biotechnol Biofuels

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BackgroundTriacylglycerols (TAGs) and wax esters (WEs) are important neutral lipids which serve as energy reservoir in some plants and microorganisms. In recent years, these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production because of the increased interest on developing renewable and environmentally benign alternatives for fossil fuels. In bacteria, the final step in TAG and WE biosynthetic pathway is catalyzed by wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT). This bifunctional WS/DGAT enzyme is also a key enzyme in biotechnological production of liquid WE via engineering of plants and microorganisms. To date, knowledge about this class of biologically and biotechnologically important enzymes is mainly from biochemical characterization of WS/DGATs from Arabidopsis, jojoba and some bacteria that can synthesize both TAGs and WEs intracellularly, whereas little is known about WS/DGATs from eukaryotic microorganisms.ResultsHere, we report the identification and characterization of two bifunctional WS/DGAT enzymes (designated TrWSD4 and TrWSD5) from the marine protist Thraustochytrium roseum. Both TrWSD4 and TrWSD5 comprise a WS-like acyl-CoA acyltransferase domain and the recombinant proteins purified from Escherichia coli Rosetta (DE3) have substantial WS and lower DGAT activity. They exhibit WS activity towards various-chain-length saturated and polyunsaturated acyl-CoAs and fatty alcohols ranging from C10 to C18. TrWSD4 displays WS activity with the lowest K m value of 0.14 μM and the highest k cat/K m value of 1.46 × 105 M−1 s−1 for lauroyl-CoA (C12:0) in the presence of 100 μM hexadecanol, while TrWSD5 exhibits WS activity with the lowest K m value of 0.96 μM and the highest k cat/K m value of 9.83 × 104 M−1 s−1 for decanoyl-CoA (C10:0) under the same reaction condition. Both WS/DGAT enzymes have the highest WS activity at 37 and 47 °C, and WS activity was greatly decreased when temperature exceeds 47 °C. TrWSD4 and TrWSD5 are insensitive to ionic strength and reduced WS activity was observed when salt concentration exceeded 800 mM. The potential of T. roseum WS/DGATs to establish novel process for biotechnological production of WEs was demonstrated by heterologous expression in recombinant yeast. Expression of either TrWSD4 or TrWSD5 in Saccharomyces cerevisiae quadruple mutant H1246, which is devoid of storage lipids, resulted in the accumulation of WEs, but not any detectable TAGs, indicating a predominant WS activity in yeast.ConclusionsThis study demonstrates both in vitro WS and DGAT activity of two T. roseum WS/DGATs, which were characterized as unspecific acyltransferases accepting a broad range of acyl-CoAs and fatty alcohols as substrates for WS activity but displaying substrate preference for medium-chain acyl-CoAs. In vivo characterization shows that these two WS/DGATs predominantly function as wax synthase and presents the feasibility for production of WEs by heterologous hosts.Electronic s...

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“…This energy is also used to remodel pyruvic acid (in the aerobic respiration) in forming of fat, proteins and other compounds. Fat then forms a layer of wax (which costs the energy) in the leaf cuticle which acts as a barrier membrane between plants and the environment which limits the exchange of water, solutes and gases (Aslan et al, 2014;Fernandez et al, 2013). Increased distribution of carbohydrate supported by high levels of boron may cause the increase of pyruvic acid and fat synthesis, which results in high wax synthesis.…”

Section: Resultsmentioning

confidence: 99%

A Study of Boron and Silica Foliar Application on Growth and Yield of Rice in High Boron Content Media

Ermawati

Agustiansyah

Pramono

et al. 2016

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Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana

Cited by 29 publications

References 49 publications

A Fatty Acyl Coenzyme A Reductase Promotes Wax Ester Accumulation in Rhodococcus jostii RHA1

A Fatty Acyl Coenzyme A Reductase Promotes Wax Ester Accumulation in Rhodococcus jostii RHA1

Two bifunctional enzymes from the marine protist Thraustochytrium roseum: biochemical characterization of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity catalyzing wax ester and triacylglycerol synthesis

A Study of Boron and Silica Foliar Application on Growth and Yield of Rice in High Boron Content Media

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