Wax synthase MhWS2 from Marinobacter hydrocarbonoclasticus: substrate specificity and biotechnological potential for wax ester production
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Wax synthases are involved in the biosynthesis of wax esters, lipids with great industrial potential. Here, we heterologously expressed the native wax synthase MhWS2 from Marinobacter hydrocarbonoclasticus in Saccharomyces cerevisiae and performed comprehensive analysis of its substrate specificity. The enzyme displayed high wax synthase (but no diacylglycerol acyltransferase) activity both in vivo and in vitro. In the presence of exogenous fatty alcohol, wax esters accounted for more than 57% of total yeast lipids. In vitro, MhWS2 produced wax esters with most of the tested substrates, showing the highest activity with 14:0-, 18:1-, 18:0-, 12:0-, and 16:0-CoA together with saturated C10-C16 fatty alcohols. Co-expression with genes encoding fatty acyl reductases resulted in the accumulation of C26-C36 wax esters. Altogether, our results provide a detailed characterization of MhWS2 which should be useful in the development of strategies for producing wax esters in various expression systems.
KeywordsWax synthase Fatty acyl reductase Wax esters Fatty alcohols Marinobacter hydrocarbonoclasticus
This work is part of ICON (Industrial Crops Producing Added Value Oils for Novel Chemicals), a European Commission-sponsored FP7 project, and was also supported by the system project “InnoDoktorant – Scholarships for PhD students, IVth edition,” co-financed by the European Union in the frame of the European Social Fund and by the Faculty of Biology, University of Gdansk (grant no. 538-L111-B593-14). GC-based analyses were performed at the Metabolome Facility of Bordeaux-MetaboHUB (ANR-11-INBS-0010).
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Conflict of interest
The authors declare that they do not have any conflicts of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Aslan S, Sun C, Leonova S, Dutta P, Dörmann P, Domergue F, Stymne S, Hofvander P (2014) Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana. Metab Eng 25:103–112. https://doi.org/10.1016/j.ymben.2014.07.001 CrossRefPubMedGoogle Scholar
- Chacón MG, Fournier AE, Tran F, Dittrich-Domergue F, Pulsifer IP, Domergue F, Rowland O (2013) Identification of amino acids conferring chain length substrate specificities on fatty alcohol-forming reductases FAR5 and FAR8 from Arabidopsis thaliana. J Biol Chem 288:30345–30355. https://doi.org/10.1074/jbc.M113.499715 CrossRefPubMedPubMedCentralGoogle Scholar
- de Jong BW, Shi S, Siewers V, Nielsen J (2014) Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway. Microb Cell Factories 13:39. https://doi.org/10.1186/1475-2859-13-39 CrossRefGoogle Scholar
- Dittrich-Domergue F, Joubès J, Moreau P, Lessire R, Stymne S, Domergue F (2014) The bifunctional protein TtFARAT from Tetrahymena thermophila catalyzes the formation of both precursors required to initiate ether lipid biosynthesis. J Biol Chem 289:21984–21994. https://doi.org/10.1074/jbc.M114.579318 CrossRefPubMedPubMedCentralGoogle Scholar
- Domergue F, Vishwanath SJ, Joubès J, Ono J, Lee JA, Bourdon M, Alhattab R, Lowe C, Pascal S, Lessire R, Rowland O (2010) Three Arabidopsis fatty acyl-coenzyme A reductases, FAR1, FAR4, and FAR5, generate primary fatty alcohols associated with suberin deposition. Plant Physiol 153:1539–1554. https://doi.org/10.1104/pp.110.158238 CrossRefPubMedPubMedCentralGoogle Scholar
- Gauthier MJ, Lafay B, Christen R, Fernandez L, Acquaviva M, Bonin P, Bertrand JC (1992) Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium. Int J Syst Bacteriol 42:568–576. https://doi.org/10.1099/00207713-42-4-568 CrossRefPubMedGoogle Scholar
- Holtzapple E, Schmidt-Dannert C (2007) Biosynthesis of isoprenoid wax ester in Marinobacter hydrocarbonoclasticus DSM 8798: identification and characterization of isoprenoid coenzyme A synthetase and wax ester synthases. J Bacteriol 189:3804–3812. https://doi.org/10.1128/JB.01932-06 CrossRefPubMedPubMedCentralGoogle Scholar
- Kalscheuer R, Stöveken T, Luftmann H, Malkus U, Reichelt R, Steinbüchel A (2006) Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters. Appl Environ Microbiol 72:1373–1379. https://doi.org/10.1128/AEM.72.2.1373-1379.2006 CrossRefPubMedPubMedCentralGoogle Scholar
- Li F, Wu X, Lam P, Bird D, Zheng H, Samuels L, Jetter R, Kunst L (2008) Identification of the wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase WSD1 required for stem wax ester biosynthesis in Arabidopsis. Plant Physiol 148:97–107. https://doi.org/10.1104/pp.108.123471 CrossRefPubMedPubMedCentralGoogle Scholar
- Röttig A, Wolf S, Steinbüchel A (2016) In vitro characterization of five bacterial WS/DGAT acyltransferases regarding the synthesis of biotechnologically relevant short-chain-length esters: test of short-chain ester synthesis by acyltransferases. Eur J Lipid Sci Technol 118:124–132. https://doi.org/10.1002/ejlt.201500200 CrossRefGoogle Scholar
- Ruiz-Lopez N, Broughton R, Usher S, Salas JJ, Haslam RP, Napier JA, Beaudoin F (2017) Tailoring the composition of novel wax esters in the seeds of transgenic Camelina sativa through systematic metabolic engineering. Plant Biotechnol J 15:837–849. https://doi.org/10.1111/pbi.12679 CrossRefPubMedPubMedCentralGoogle Scholar
- Sheng J, Feng X (2015) Metabolic engineering of yeast to produce fatty acid-derived biofuels: bottlenecks and solutions. Front Microbiol 6. https://doi.org/10.3389/fmicb.2015.00554
- Shi S, Valle-Rodríguez JO, Khoomrung S, Siewers V, Nielsen J (2012) Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production. Biotechnol Biofuels 5:7. https://doi.org/10.1186/1754-6834-5-7 CrossRefPubMedPubMedCentralGoogle Scholar
- Shi S, Valle-Rodríguez JO, Siewers V, Nielsen J (2014) Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters: enhancement of FAEEs production. Biotechnol Bioeng 111:1740–1747. https://doi.org/10.1002/bit.25234 CrossRefPubMedGoogle Scholar
- Stanley DW, Nelson DR (eds) (1993) Insect lipids: chemistry, biochemistry, and biology. University of Nebraska Press, LincolnGoogle Scholar
- Stöveken T, Kalscheuer R, Malkus U, Reichelt R, Steinbüchel A (2005) The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol 187:1369–1376. https://doi.org/10.1128/JB.187.4.1369-1376.2005 CrossRefPubMedPubMedCentralGoogle Scholar
- Subileau M, Jan A-H, Nozac’h H, Pérez-Gordo M, Perrier V, Dubreucq E (2015) The 3D model of the lipase/acyltransferase from Candida parapsilosis, a tool for the elucidation of structural determinants in CAL-A lipase superfamily. Biochim Biophys Acta BBA - Proteins Proteomics 1854:1400–1411. https://doi.org/10.1016/j.bbapap.2015.06.012 CrossRefPubMedGoogle Scholar
- Valle-Rodríguez JO, Shi S, Siewers V, Nielsen J (2014) Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid ethyl esters, an advanced biofuel, by eliminating non-essential fatty acid utilization pathways. Appl Energy 115:226–232. https://doi.org/10.1016/j.apenergy.2013.10.003 CrossRefGoogle Scholar
- Villa JA, Cabezas M, de la Cruz F, Moncalián G (2014) Use of limited proteolysis and mutagenesis to identify folding domains and sequence motifs critical for wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase activity. Appl Environ Microbiol 80:1132–1141. https://doi.org/10.1128/AEM.03433-13 CrossRefPubMedPubMedCentralGoogle Scholar
- Wagner M, Hoppe K, Czabany T, Heilmann M, Daum G, Feussner I, Fulda M (2010) Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri. Plant Physiol Biochem PPB Société Fr Physiol Végétale 48:407–416. https://doi.org/10.1016/j.plaphy.2010.03.008 CrossRefGoogle Scholar
- Zhu L-H, Krens F, Smith MA, Li X, Qi W, van Loo EN, Iven T, Feussner I, Nazarenus TJ, Huai D, Taylor DC, Zhou X-R, Green AG, Shockey J, Klasson KT, Mullen RT, Huang B, Dyer JM, Cahoon EB (2016) Dedicated industrial oilseed crops as metabolic engineering platforms for sustainable industrial feedstock production. Sci Rep 6:22181. https://doi.org/10.1038/srep22181 CrossRefPubMedPubMedCentralGoogle Scholar