Abstract
Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.
Similar content being viewed by others
References
Aichholz R, Lorbeer E (2000) Investigation of combwax of honeybees with high-temperature gas chromatography and high-temperature gas chromatography-chemical ionization mass spectrometry II: high-temperature gas chromatography-chemical ionization mass spectrometry. J Chromatogr A 883:75–88. doi:10.1016/s0021-9673(00)00386-1
Aslan S et al (2014) Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana. Metab Eng 25C:103–112. doi:10.1016/j.ymben.2014.07.001
Barney BM, Wahlen BD, Garner E, Wei J, Seefeldt LC (2012) Differences in substrate specificities of five bacterial wax ester synthases. Appl Environ Microbiol 78:5734–5745. doi:10.1128/AEM.00534-12
Carlsson AS, Yilmaz JL, Green AG, Stymne S, Hofvander P (2011) Replacing fossil oil with fresh oil—with what and for what? Eur J Lipid Sci Technol 113:812–831. doi:10.1002/ejlt.201100032
Chapman KD, Dyer JM, Mullen RT (2013) Commentary: why don’t plant leaves get fat? Plant Sci Int J Exp Plant Biol 207:128–134. doi:10.1016/j.plantsci.2013.03.003
Cheng JB, Russell DW (2004) Mammalian wax biosynthesis. I. Identification of two fatty acyl-Coenzyme A reductases with different substrate specificities and tissue distributions. J Biol Chem 279:37789–37797. doi:10.1074/jbc.M406225200
Durrett TP, Benning C, Ohlrogge J (2008) Plant triacylglycerols as feedstocks for the production of biofuels. Plant J Cell Mol Biol 54:593–607. doi:10.1111/j.1365-313X.2008.03442.x
Fixter LM, Nagi MN, McCormack JG, Fewson CA (1986) Structure, distribution and function of wax esters in acinetobacter-calcoaceticus. J Gen Microbiol 132:3147–3157
Heilmann M, Iven T, Ahmann K, Hornung E, Stymne S, Feussner I (2012) Production of wax esters in plant seed oils by oleosomal cotargeting of biosynthetic enzymes. J Lipid Res 53:2153–2161. doi:10.1194/jlr.M029512
Hofvander P, Doan TT, Hamberg M (2011) A prokaryotic acyl-CoA reductase performing reduction of fatty acyl-CoA to fatty alcohol. FEBS Lett 585:3538–3543. doi:10.1016/j.febslet.2011.10.016
Horsch RB, Klee HJ, Stachel S, Winans SC, Nester EW, Rogers SG, Fraley RT (1986) Analysis of agrobacterium-tumefaciens virulence mutants in leaf-disks. Proc Natl Acad Sci USA 83:2571–2575. doi:10.1073/pnas.83.8.2571
Ishige T, Tani A, Sakai Y, Kato N (2003) Wax ester production by bacteria. Curr Opin Microbiol 6:244–250. doi:10.1016/s1369-5274(03)00053-5
Kalscheuer R, Steinbuchel A (2003) A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278:8075–8082. doi:10.1074/jbc.M210533200
Khan AA, Kolattuk P (1973) Control of synthesis and distribution of acyl moieties in etiolated euglena-gracilis. Biochemistry 12:1939–1948. doi:10.1021/bi00734a017
Kolattukudy PE, Rogers L (1978) Biosynthesis of fatty alcohols, alkane-1,2-diols and wax esters in particulate preparations from uropygial glands of white-crowned sparrows (zonotrichia-leucophrys). Arch Biochem Biophys 191:244–258. doi:10.1016/0003-9861(78)90087-5
Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42:51–80. doi:10.1016/s0163-7827(02)00045-0
Lardizabal KD, Metz JG, Sakamoto T, Hutton WC, Pollard MR, Lassner MW (2000) Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic Arabidopsis. Plant Physiol 122:645–655. doi:10.1104/pp.122.3.645
Li W et al (2010) Green waxes, adhesives and lubricants. Philos Trans Ser A Math Phys Eng Sci 368:4869–4890. doi:10.1098/rsta.2010.0197
Liu D, Shi L, Han C, Yu J, Li D, Zhang Y (2012) Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR. PLoS ONE 7:e46451. doi:10.1371/journal.pone.0046451
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408. doi:10.1006/meth.2001.1262
Metz JG, Pollard MR, Anderson L, Hayes TR, Lassner MW (2000) Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed. Plant Physiol 122:635–644. doi:10.1104/pp.122.3.635
Miwa TK (1971) Jojoba oil wax esters and derived fatty acids and alcohols: gas chromatographic analyses. J Am Oil Chem Soc 48:259–264. doi:10.1007/bf02638458
Moto K et al (2003) Pheromone gland-specific fatty-acyl reductase of the silkmoth, Bombyx mori. Proc Natl Acad Sci USA 100:9156–9161. doi:10.1073/pnas.1531993100
PostBeittenmiller D (1996) Biochemistry and molecular biology of wax production in plants. Ann Rev Plant Physiol Plant Mol Biol 47:405–430. doi:10.1146/annurev.arplant.47.1.405
Reiser S, Somerville C (1997) Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzyme a reductase. J Bacteriol 179:2969–2975
Samuels L, Kunst L, Jetter R (2008) Sealing plant surfaces: cuticular wax formation by epidermal cells. Annu Rev Plant Biol 59:683–707. doi:10.1146/annurev.arplant.59.103006.093219
Sturaro M, Hartings H, Schmelzer E, Velasco R, Salamini F, Motto M (2005) Cloning and characterization of GLOSSY1, a maize gene involved in cuticle membrane and wax production. Plant Physiol 138:478–489. doi:10.1104/pp.104.058164
Vanhercke T, Wood CC, Stymne S, Singh SP, Green AG (2013) Metabolic engineering of plant oils and waxes for use as industrial feedstocks. Plant Biotechnol J 11:197–210. doi:10.1111/pbi.12023
Wahlen BD, Oswald WS, Seefeldt LC, Barney BM (2009) Purification, characterization, and potential bacterial wax production role of an NADPH-dependent fatty aldehyde reductase from Marinobacter aquaeolei VT8. Appl Environ Microbiol 75:2758–2764. doi:10.1128/AEM.02578-08
Acknowledgments
We thank Prof. Sten Stymne (Dept. of Plant Breeding, SLU, Alnarp) for support and advice, and Drs. Sarosh Bejai (Dept. of Plant Biology, SLU, Uppsala) and Frédéric Domergue (Laboratoire de Biogenèse Membranaire, CNRS, Univ. Bordeaux Ségalen, Bordeaux, France) for advice concerning QPCR data analysis and DNA constructions, respectively. The work was supported by EU FP7 project “ICON”, the Swedish Research Council Formas, and the Swedish Governmental Agency for Innovation Systems, VINNOVA.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare they have no competing interests.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Aslan, S., Hofvander, P., Dutta, P. et al. Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion. Transgenic Res 24, 945–953 (2015). https://doi.org/10.1007/s11248-015-9893-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-015-9893-5