Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters

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.     

Biosynthesis of wax esters in tissues of Sinapis alba L. seeds

The biosynthesis of wax esters has been investigated in maturing seeds of Sinapis alba. Exogenous long-chain alcohols are incorporated exclusively into alkyl moieties of wax esters. Oxidation of the long-chain alcohols is not detected. Exogenous fatty acids are incorporated into acyl moieties of wax esters to a low extent. A reduction of fatty acids to alcohols is not observed. Synthesis of wax esters is localized exclusively in the testa; both outer and inner integument are equally active in wax ester biosynthesis. The biosynthesis of wax esters is specific with regard to both chain length and degree of unsaturation of long-chain alcohols. Exogenous and endogenous sterols are not esterified.     

Synthesis of Novel Lipids in Saccharomyces cerevisiae by Heterologous Expression of an Unspecific Bacterial Acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

Oleyl Oleate and Homologous Wax Esters Synthesized Coordinately from Oleic Acid by Acinetobacter and Coryneform Strains

Newly isolated Acinetobacter (NRRL B-14920, B-14921, B-14923) and coryneform (NRRL B-14922) strains accumulated oleyl oleate and homologous liquid wax esters (C_30:2–C_36:2) in culture broths. Diunsaturated oleyl oleate preponderated in 75 mg liquid wax esters (280 mg lipid extract) recovered from 100-ml cultures of Acinetobacter B-14920 supplemented with 810 mg oleic acid–oleyl alcohol. With soybean oil instead of oleic acid, wax esters (260 mg) were increased to approximately 50% of the lipid extract. Production of wax esters by cultures supplemented with combined fatty (C₈–C₁₈) alcohols and acids suggests a coordinated synthesis whereby the exogenous alcohol remains unaltered, and the fatty acid is partially oxidized with removal of C₂ units before esterification. Consequently, C₈–C₁₈ primary alcohols control chain lengths of the wax esters. Exogenous fatty acids are presumed to enter an intracellular oxidation pool from which is produced a homologous series of liquid wax esters.     

The composition of external lipids from adult whiteflies,Bemisia tabaci and Trialeurodes vaporariorum

The total surface lipids, including the wax particles, of the adult whiteflies of Bemisia tabaci and Trialeurodes vaporariorum were characterized. At eclosion, there were similar amounts of long-chain hydrocarbons, aldehydes, alcohols and wax esters. Within a few hours post-eclosion, long-chain aldehydes and long-chain alcohols were the dominant surface lipid components, C₃₄ on B. tabaci and C₃₂ on T. vaporariorum. Hydrocarbons, mainly n-alkanes, were minor components of the surface lipids. The major wax esters were C₄₆ on B. tabaci and C₄₂ on T. vaporariorum. The major acid and alcohol moieties in the wax esters of B. tabaci were C₂₀ and C₂₆, respectively, and of T. vaporariorum were C₂₀ and C₂₂, respectively. Both B. tabaci and T. vaporariorum had a minor wax ester composed of the fatty acid C_18:1 esterified to the major alcohols, C₃₄ and C₃₂, respectively. Bemisia were readily distinguished from Trialeurodes based on the composition of their wax particles and/or their wax esters; however, no differentiating surface lipid components were detected between biotypes A and B of B. tabaci.     

Assessment of bacterial acyltransferases for an efficient lipid production in metabolically engineered strains of E. coli

Microbially produced lipids like triacylglycerols or fatty acid ethyl esters are currently of great interest as fuel replacements or other industrially relevant compounds. They can even be produced by non-oleaginous microbes, like Escherichia coli, upon metabolic engineering. However, there is still much room for improvement regarding the yield for a competitive microbial production of lipids or biofuels. We genetically engineered E. coli by expressing fadD, fadR, pgpB, plsB and ‘tesA in combination with atfA from Acinetobacter baylyi. A total fatty acid contents of up to 16% (w/w) was obtained on complex media, corresponding to approximately 9% (w/w) triacylglycerols and representing the highest titers of fatty acids and triacylglycerols obtained in E. coli under comparable cultivation conditions, so far. To evaluate further possibilities for an optimization of lipid production, ten promising bacterial wax ester synthase/acyl-Coenzyme A:diacylglycerol acyltransferases were tested and compared. While highest triacylglycerol storage was achieved with AtfA, the mutated variant AtfA-G355I turned out to be most suitable for fatty acid ethyl ester biosynthesis and enabled an accumulation of approx. 500 mg/L without external ethanol supplementation.     

A Comparison of the Composition of Wax Ester Molecular Species of Different Coral Groups (Subclasses Hexacorallia and Octocorallia)

Wax esters, which are esters of fatty alcohols and fatty acids (FAs), are one of the main classes of reserve lipids in all coral species. The chemical structures and the content of wax ester molecular species were determined for the first time in nine coral species from three taxonomic groups: symbiotic reef-building corals, (Hexacorallia subclasses), symbiotic soft coral alcyonarians, and asymbiotic soft coral gorgonians (Octocorallia subclasses) collected in the South China Sea (Vietnam). Our comparison of these groups showed that the absence of symbiotic microalgae (zooxanthellae) and the exoskeleton affects the profile of molecular species of wax esters considerably. The main components of wax esters of all corals were cetyl palmitate (16:0-16:0) and other saturated wax esters containing 30, 34, and 36 carbon atoms. The content of unsaturated molecular species 6:0–16:1, 16:0–18:1, and 16:0–20:1 in wax esters of symbiotic soft corals (alcyonarians) was greater than that in wax esters of reef-building corals. In contrast to symbiotic coral species, wax esters of asymbiotic soft corals, namely azooxanthellate gorgonians, contained a considerable amount of long-chain molecular species (C₃₇-C₄₁) with an odd number of carbon atoms. The presence of such molecular species indicates that asymbiotic gorgonians may use bacterial FAs in biosynthesis of their own wax esters. This observation confirms our hypothesis that bacterial community is important for maintaining the energy balance of azooxanthellate corals.     

Fatty acid alcohol ester-synthesizing activity of lipoprotein lipase

The fatty acid alcohol ester-synthesizing activity of lipoprotein lipase (LPL) was characterized using bovine milk LPL. Synthesizing activities were determined in an aqueous medium using oleic acid or trioleylglycerol as the acyl donor and equimolar amounts of long-chain alcohols as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with LPL, palmityl oleate was synthesized, in a time- and dose-dependent manner. Apo-very low density lipoprotein (apoVLDL) stimulated LPL-catalyzed palmityl oleate synthesis. The apparent equilibrium ratio of fatty acid alcohol ester/oleic acid was estimated using a high concentration of LPL and a long (20 h) incubation period. The equilibrium ratio was affected by the incubation pH and the alcohol chain length. When the incubation pH was below pH 7.0 and long chain fatty acyl alcohols were used as substrates, the fatty acid alcohol ester/free fatty acid equilibrium ratio favored ester formation, with an apparent equilibrium ratio of fatty acid alcohol ester/fatty acid of about 0.9/0.1. The equilibrium ratio decreased sharply at alkaline pH (above pH 8.0). The ratio also decreased when fatty alcohols with acyl chains shorter than dodecanol were used. When a trioleoylglycerol/fatty acyl alcohol emulsion was incubated with LPL, fatty acid alcohol esters were synthesized in a dose- and time-dependent fashion. Fatty acid alcohol esters were easily synthesized from trioleoylglycerol when fatty alcohols with acyl chains longer than dodecanol were used, but synthesis was decreased with fatty alcohols with acyl chain lengths shorter than decanol, and little synthesizing activity was detected with shorter-chain fatty alcohols such as butanol or ethanol.     

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

In a future bio-based economy, renewable sources for lipid compounds at attractive cost are needed for applications where today petrochemical derivatives are dominating. Wax esters and fatty alcohols provide diverse industrial uses, such as in lubricant and surfactant production. In this study, chloroplast metabolism was engineered to divert intermediates from de novo fatty acid biosynthesis to wax ester synthesis. To accomplish this, chloroplast targeted fatty acyl reductases (FAR) and wax ester synthases (WS) were transiently expressed in Nicotiana benthamiana leaves. Wax esters of different qualities and quantities were produced providing insights to the properties and interaction of the individual enzymes used. In particular, a phytyl ester synthase was found to be a premium candidate for medium chain wax ester synthesis. Catalytic activities of FAR and WS were also expressed as a fusion protein and determined functionally equivalent to the expression of individual enzymes for wax ester synthesis in chloroplasts.     

Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed

The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve. This is in contrast to the triglycerides found in seeds of other plants. We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme. Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols. The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development. When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils. Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production. In addition to free alcohols, novel wax esters were detected in the transgenic seed oils. In vitro assays revealed that B. napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis. Thus, introduction of a single cDNA into B. napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes.     

Production of wax esters in plant seed oils by oleosomal cotargeting of biosynthetic enzymes

Wax esters are neutral lipids exhibiting desirable properties for lubrication. Natural sources have traditionally been whales. Additionally some plants produce wax esters in their seed oil. Currently there is no biological source available for long chain length monounsaturated wax esters that are most suited for industrial applications. This study aimed to identify enzymatic requirements enabling their production in oilseed plants. Wax esters are generated by the action of fatty acyl-CoA reductase (FAR), generating fatty alcohols and wax synthases (WS) that esterify fatty alcohols and acyl-CoAs to wax esters. Based on their substrate preference, a FAR and a WS from Mus musculus were selected for this study (MmFAR1 and MmWS). MmWS resides in the endoplasmic reticulum (ER), whereas MmFAR1 associates with peroxisomes. The elimination of a targeting signal and the fusion to an oil body protein yielded variants of MmFAR1 and MmWS that were cotargeted and enabled wax ester production when coexpressed in yeast or Arabidopsis. In the fae1 fad2 double mutant, rich in oleate, the cotargeted variants of MmFAR1 and MmWS enabled formation of wax esters containing >65% oleyl-oleate. The data suggest that cotargeting of unusual biosynthetic enzymes can result in functional interplay of heterologous partners in transgenic plants.     

A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1

Triacylglycerols (TAGs) and wax esters are neutral lipids with considerable importance for dietetic, technical, cosmetic, and pharmaceutical applications. Acinetobacter calcoaceticus ADP1 accumulates wax esters and TAGs as intracellular storage lipids. We describe here the identification of a bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS) as well as acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. Experiments with a knock-out mutant demonstrated the key role of the bifunctional WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA acyltransferase is not related to known acyltransferases including the WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2 families present in yeast, plants, and animals, and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in yeast and plants. A large number of WS/DGAT-related proteins were identified in Mycobacterium and Arabidopsis thaliana indicating an important function of these proteins. WS and DGAT activity was demonstrated for the translational product of one WS/DGAT homologous gene from M. smegmatis mc(2)155. The potential of WS/DGAT to establish novel processes for biotechnological production of jojoba-like wax esters was demonstrated by heterologous expression in recombinant Pseudomonas citronellolis. The potential of WS/DGAT as a selective therapeutic target of mycobacterial infections is discussed.     

Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

The lipid biochemistry of calanoid copepods

Calanus species, particularly those in high latitudes, can accumulate large oil reserves consisting predominantly of wax esters. These wax esters consist predominantly of 16:0, 20:1 (n–9) and 22:1 (n–11) fatty alcohols, mainly formed de novo by the animals from non-lipid dietary precursors, esterified with various fatty acids that are often polyunsaturated fatty acids and largely of dietary, phytoplanktonic origin. Wax ester formation is maximal in copepodite stages IV and V. The lipids are elaborated not primarily for buoyancy regulation but as a source of metabolic energy during overwintering, particularly for reproduction. Large quantities of wax esters are utilised for gonadal development when stage V copepodites mature to females. Development of stage V copepodites to males is not accompanied by wax ester utilisation but males consume large amounts of these lipids in physical activity during reproduction. The role of wax esters in the life history of calanoids is illustrated with particular reference to a comparison of Calanus finmarchicus and Metridia longa in Balsfjord, northern Norway.     

Phase Behaviour and Formation of Fatty Acid Esters Nanoemulsions Containing Piroxicam

Fatty acid esters are long-chain esters, produced from the reaction of fatty acids and alcohols. They possess potential applications in cosmetic and pharmaceutical formulations due to their excellent wetting behaviour at interfaces and a non-greasy feeling when applied on the skin surfaces. This preliminary work was carried out to construct pseudo-ternary phase diagrams for oleyl laurate, oleyl stearate and oleyl oleate with surfactants and piroxicam. Then, the preparation and optimization study via ‘One-At-A-Time Approach’ were carried out to determine the optimum amount of oil, surfactants and stabilizer using low-energy emulsification method. The results revealed that multi-phase region dominated the three pseudo-ternary phase diagrams. A composition was chosen from each multi-phase region for preparing the nanoemulsions systems containing piroxicam by incorporating a hydrocolloid stabilizer. The results showed that the optimum amount (w/w) of oil for oleyl laurate nanoemulsions was 30 and 20 g (w/w) for oleyl stearate nanoemulsions and oleyl oleate nanoemulsions. For each nanoemulsions system, the amount of mixed surfactants and stabilizer needed for the emulsification to take place was found to be 10 and 0.5 g (w/w), respectively. The emulsification process via high-energy emulsification method successfully produced nano-sized range particles. The nanoemulsions systems passed the centrifugation test and freeze–thaw cycle with no phase failures, and stable for 3 months at various storage temperatures (3°C, 25°C and 45°C). The results proved that the prepared nanoemulsions system cannot be formed spontaneously, and thus, energy input was required to produce nano-sized range particles.     

Synthesis of wax esters by a cell-free system from barley (Hordeum vulgare L.)

Experimental evidence for a membranebound microsomal ester synthetase from Bonus barley primary leaves is reported. The results are consistent with at least two mechanisms for the synthesis of barley wax esters: an acyl-CoA-fattyalcohol-transacylase-type reaction and an apparent direct esterification of alcohols with fatty acids. Biosynthesis of wax esters was not specific with regard to the chain length of the tested alcohols. The microsomal preparation readily catalyzed the esterification of C₁₆-, C₁₈-, C₂₂- or C₂₄-labelled alcohols with fatty acids of endogenous origin. Exogenous long-chain alcohols were exclusively incorporated into the alkyl moieties of the esters. Addition of ATP, CoA and-or free fatty acids was not effective in stimulating or depressing the esterifying activity of the microsomal fraction. Partial solubilization of the ester synthetase was obtained using phosphate-buffered saline.Abbreviations P pellet - PBS phosphate-buffered saline - S supernatant - SDS sodium dodecyl sulphate     

Waxes and lipids associated with the external waxy structures of nymphs and pupae of the giant whitefly, Aleurodicus dugesii

The nymphs and pupae of the giant whitefly, Aleurodicus dugesii, produce large quantities of external lipids, both as waxy particles and as waxy filaments. The nymphs and pupae extrude filaments from two dorsal rows of five pores each. Filaments can attain lengths of 5-8 cm. The external lipids of nymphs and pupae consist largely of long-chain aldehydes, alcohols, acetate esters and wax esters. Hydrocarbons are minor components. Soon after hatching, the nymph produced an unidentified waxy fringe extruded laterally from its margin. After molting to the second instar, long, hollow, waxy filaments were produced by the immature stages. The major lipid class associated with the filaments was saturated wax esters (89%), mainly C44, C46 and C60. Associated with formation of the filaments were waxy particles in the shape of curls, which peeled off of the extruding filaments. Similar but more tubular-shaped curls were also produced by numerous lateral pores so that, eventually, the curls completely camouflaged the nymph. The major lipid class of the curls was wax esters (50%), mainly C44 and C46. The cuticular surface lipids of the nymphs were mainly long-chain aldehydes (43%) and wax esters (27%). Unsaturated fatty acid moieties constituted 2 and 19% of the wax esters of curls and nymph cuticular surface lipids, respectively. The major lipid classes of pupae and of their palisade were long-chain aldehydes and alcohols. No unsaturated wax esters were detected in the filaments, but 30% of pupal and 21% of palisade surface wax esters were unsaturated in their fatty acid moieties, 16:1, 18:1 and 20:1.     

The surface wax composition of the exuviae and adults of Aleyrodes singularis

Long-chain aldehydes, alcohols, hydrocarbons and wax esters were major components of the external lipids of adult Aleyrodes singularis. In exuviae, acetate esters replaced the hydrocarbons as a major component. The major long-chain alcohol and aldehyde from adults were C32 and were essentially the exclusive components of the wax particles. The major alcohol from exuviae was C26 and the aldehydes were C26, C28, C30 and C32. The major acetate esters were C28 and C30 in both adults and exuviae. There were wax esters of similar carbon number in adults and exuviae although the exuviae had a greater amount of wax esters with unsaturated fatty acids. The fatty acid and alcohol composition of the wax esters differed markedly between adults and exuviae. Wax esters of adults had similar amounts of C16, C18, C20, C22 and C24 fatty acids while those from exuviae contained largely C16 and C18. The major alcohol in the wax esters of adults was C22 and those of exuviae were C26 and C28. The distribution of fatty acids and alcohols among wax esters of varying chain length also differed between adults and exuviae: in adults C22 was the major fatty acid found in the dominant wax ester, C44 and the C22 alcohol was the major alcohol and found in wax esters C42 and C44. In exuviae C16 and C18 were the major fatty acids found in most wax esters and a C28 alcohol was the major alcohol found in wax esters C44 and C46, the two dominant wax esters in exuviae. It was clear that the difference in chemistry of the wax esters between the adults and exuviae is not evident unless the acid and alcohol moieties are characterized.     

Preparation and Evaluation of Jojoba Oil Methyl Esters as Biodiesel and as a Blend Component in Ultra-Low Sulfur Diesel Fuel

The jojoba plant (Simmondsia chinensis L.) produces seeds that contain around 50 to 60 wt.% of inedible long-chain wax esters that are suitable as a potential feedstock for biodiesel (BD) production. Jojoba oil methyl esters (JME) were prepared from acid-catalyzed pretreated jojoba oil in order to evaluate important fuel properties of jojoba-based BD, including kinematic viscosity, cloud point (CP), pour point (PP), cold filter plugging point (CFPP), acid value (AV), oxidative stability, and lubricity. A comparison was made with soybean oil methyl esters (SME) and relevant BD fuel standards such as ASTM D6751 and EN 14214. JME was characterized using Fourier transform infrared spectroscopy and ¹H and ¹³C nuclear magnetic resonance. The CP, PP, and CFPP of JME were ?13°C, ?16°C, and ?14°C, respectively, which were superior to SME. The kinematic viscosity (40°C) of JME was 6.67 mm²/s, which was higher than observed for SME. Blends (B5 and B20) of JME in ultra-low sulfur diesel fuel (ULSD) were also evaluated for the aforementioned fuel properties and compared to an analogous set of blends of SME in ULSD and relevant petro diesel fuel standards such as ASTM D975 and D7467. JME blends in ULSD displayed improved low-temperature properties in comparison to neat ULSD and blends of SME in ULSD. In summary, jojoba oil has potential as an alternative, nonfood feedstock for BD production.