scholarly journals Plastidial Wax Ester Biosynthesis As A Tool To Synthesize Shorter And More Saturated Wax Esters

2021 ◽  
Author(s):  
Katharina Vollheyde ◽  
Ellen Hornung ◽  
Cornelia Herrfurth ◽  
Till Ischebeck ◽  
Ivo Feussner
Keyword(s):  
Transgenic Plants ◽  
Fossil Fuel ◽  
Neutral Lipids ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Wax Ester ◽  
In Planta ◽  
Alternative Source ◽  
Transit Peptides ◽  
Ester Biosynthesis

Abstract Background: Wax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings and cosmetics. They can be produced chemically from fossil fuel or plant derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation degree of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, a tightly controlled tailor-made WE synthesis in plants is aimed for. Here, we report the generation of ten combinations of WE producing transgenes expressed in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and synthesized WE species in the transgenic plants.Results: The transgenes consisted of different combinations of a fatty acyl-CoA/ACP reductase (FAR) and two wax synthases/acyl-CoA:diacylglycerol O-acyltransferases (WSD) namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access diverse alcohol and acyl substrate pools within A. thaliana. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis.Conclusions: A shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE mainly derived from mid and long chain saturated and monounsaturated substrates.

scholarly journals Plastidial wax ester biosynthesis as a tool to synthesize shorter and more saturated wax esters

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Katharina Vollheyde ◽  
Ellen Hornung ◽  
Cornelia Herrfurth ◽  
Till Ischebeck ◽  
Ivo Feussner
Keyword(s):  
Transgenic Plants ◽  
Fossil Fuel ◽  
Neutral Lipids ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Wax Ester ◽  
In Planta ◽  
Alternative Source ◽  
Transit Peptides ◽  
Ester Biosynthesis

Abstract Background Wax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings, and cosmetics. They can be produced chemically from fossil fuel or plant-derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, tightly controlled and tailor-made WE synthesis in plants would be a sustainable, beneficial, and valuable commodity. Here, we report the expression of ten combinations of WE producing transgenes in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and composition in the transgenic plants. Results The transgenes consisted of different combinations of a FATTY ACYL-COA/ACP REDUCTASE (FAR) and two WAX SYNTHASES/ACYL-COA:DIACYLGLYCEROL O-ACYLTRANSFERASES (WSD), namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access distinct alcohol and acyl substrate pools within A. thaliana cells. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis. Conclusions A shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE that are mainly derived from mid- and long-chain saturated and monounsaturated substrates.

scholarly journals Fatty Alcohols for Wax Esters in Marinobacter aquaeolei VT8: Two Optional Routes in the Wax Biosynthesis Pathway

2013 ◽  
Vol 79 (22) ◽  
pp. 7055-7062 ◽  
Author(s):  
Eric M. Lenneman ◽  
Janet M. Ohlert ◽  
Nagendra P. Palani ◽  
Brett M. Barney
Keyword(s):  
Fatty Alcohol ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Transcriptional Analysis ◽  
Wax Ester ◽  
Fatty Aldehyde ◽  
Marinobacter Aquaeolei

ABSTRACTThe biosynthesis of wax esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previousin vitroenzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol inMarinobacter aquaeoleiVT8. 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 biosynthesisin 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 previousin vitrostudies and provide the first glimpse into the role of each enzymein vivoin the native organism.

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

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
James Round ◽  
Raphael Roccor ◽  
Shu-Nan Li ◽  
Lindsay D. Eltis
Keyword(s):  
Specific Activity ◽  
Dry Weight ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Wax Ester ◽  
Wild Type ◽  
Considerable Potential ◽  
Commodity Chemicals ◽  
Acyl Coenzyme A ◽  
Rhodococcus Jostii

ABSTRACT 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 ± 3 nmol/mg · min and dodecanal to dodecanol with a specific activity of 5,300 ± 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 ∼13% cellular dry weight under lipid-accumulating conditions, and their acyl moieties had longer average chain lengths than those in wild-type cells (C17 versus C16). The results provide insight into the biosynthesis of WEs in rhodococci and facilitate the development of this genus for the production of high-value 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.

scholarly journals Neutral Lipid Biosynthesis in Engineered Escherichia coli: Jojoba Oil-Like Wax Esters and Fatty Acid Butyl Esters

2006 ◽  
Vol 72 (2) ◽  
pp. 1373-1379 ◽  
Author(s):  
Rainer Kalscheuer ◽  
Tim Stöveken ◽  
Heinrich Luftmann ◽  
Ursula Malkus ◽  
Rudolf Reichelt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Neutral Lipids ◽  
Wax Esters ◽  
Wax Ester ◽  
Jojoba Oil ◽  
E Coli ◽  
Butyl Esters ◽  
Long Chain

ABSTRACT Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.

scholarly journals Wax ester production in nitrogen-rich conditions by metabolically engineered Acinetobacter baylyi ADP1

2019 ◽  
Author(s):  
Jin Luo ◽  
Elena Efimova ◽  
Pauli Losoi ◽  
Ville Santala ◽  
Suvi Santala
Keyword(s):  
Metabolic Engineering ◽  
Isocitrate Lyase ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Wax Ester ◽  
High Carbon ◽  
Acinetobacter Baylyi ◽  
Storage Lipids ◽  
Carbon Nitrogen Ratio ◽  
Nitrogen Ratio

AbstractMetabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal. An example of a synthesis pathway strongly dependent on external conditions is the production of storage lipids, which typically requires high carbon/nitrogen ratio. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered the central carbon metabolism of A. baylyi ADP1 by deletion of the gene aceA encoding for isocitrate lyase in order to allow redirection of carbon towards WEs. The production was further enhanced by overexpression of fatty acyl-CoA reductase Acr1 in the wax ester production pathway. 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.

scholarly journals Do minke whales (Balaenoptera acutorostrata) digest wax esters?

1995 ◽  
Vol 74 (5) ◽  
pp. 717-722 ◽  
Author(s):  
Erling S. Nordøy
Keyword(s):  
Energy Density ◽  
Dry Matter ◽  
General Pattern ◽  
Dry Mass ◽  
Wax Esters ◽  
Gut Contents ◽  
Wax Ester ◽  
Balaenoptera Acutorostrata ◽  
Minke Whales

Mammals are known to utilize wax esters with an efficiency of less than 50%. The purpose of the present study was to examine whether or not minke whales (Balaenoptera acutorostrata), which at times may eat considerable amounts of wax-ester-rich krill, represent an exception to this general pattern. Samples of fresh undigested forestomach, as well as colon, contents were obtained from minke whales (n5) that had been feeding on krill (Thysanoessa inermis) for some time. The samples were analysed for dry mass, energy density, lipid content and the major lipid classes, including wax esters. The concentrations of wax esters were compared with previous estimates of dry-matter disappearance of the same type of prey using anin vitrotechnique, to calculate the dry-matter digestibility of wax esters (DMDwax). Wax esters contributed 21% of the energy and 47% of total lipids in the krill diet. The energy density of gut contents decreased by 50% after their passage from forestomach to the end of the colon. The DMDwaxwas 94·1 (SD 2·8)% (n5). This high DMDwaxand the occurrence of fatty alcohols, one of the products of wax-ester hydrolysis, in faeces show that minke whales are very efficient digesters of wax esters and absorb most of the energy-rich products of this process.

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 Optimization of Agrobacterium-mediated transformation conditions of prospective genotypes of winter bread wheat by in planta method

2021 ◽  
Vol 28 ◽  
pp. 66-71
Author(s):  
O. V. Dubrovna ◽  
L. V. Slivka
Keyword(s):  
Transgenic Plants ◽  
Bread Wheat ◽  
Optical Density ◽  
Dna Transfer ◽  
Plant Genome ◽  
In Planta ◽  
Agrobacterium Mediated Transformation ◽  
The Third ◽  
Winter Bread Wheat ◽  
Inoculation Medium

Aim. Optimization of conditions for genetic transformation of new promising genotypes of winter bread wheat (T. aestivum L.) by in planta method. Methods. Agrobacterium-mediated transformation by in planta method using the strain AGL0 and vector construct pBi2E. Results. The influence of air temperature, optical density of cells of agrobacterial suspension, inoculation day and composition of inoculation medium on the frequency of obtaining transgenic plants of new winter wheat genotypes was studied. The dependence of the frequency obtaining of transgenic plants from environmental conditions, in particular temperature, has been established. It was found that the temperature regime of 20-22°C provided the largest number (4.8%) of wheat transformants, and when the temperature is reduced to 16-18°C there is a decrease in the efficiency of T-DNA transfer into the plant genome and the lowest frequency of transformation (0.7%). Conclusions. The largest number of transformants was obtained using a inoculation medium without sucrose, the optical density of cells of the agrobacterial suspension of 0.6 op.od. and inoculation on the third day after castration of ears. Keywords: T. aestivum, Agrobacterium-mediated transformation in planta, optimization of conditions.

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