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Identification and characterization of a long-chain fatty acid transporter in the sophorolipid-producing strain Starmerella bombicola

  • Applied genetics and molecular biotechnology
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Abstract

The sophorolipid-producing strain Starmerella bombicola CGMCC 1576 has a remarkable ability to produce sophorolipids (SLs) under the acidic and lactonic forms with almost equal proportion. In this study, we found the gene encoding for the long-chain acyl-CoA synthetase (ALCS). This enzyme was putatively identified as a membrane-bound long-chain fatty acid transport protein and contributed to the uptake of long-chain fatty acids. Disruption of the alcs gene resulted in an impaired growth of the alcs-deleted mutant in minimal media containing different fatty acids (C12:0, C14:0, C16:0, C18:0, C22:0, and C24:0) as the sole carbon source and led to a dramatic decrease in the uptake of the fluorescent-tagged long-chain fatty acid analogue—boron dipyrromethene difluoride dodecanoic acid (BODIPY-3823). The absence of this alcs gene caused obvious phenotype changes. Compared with the wild-type strain, the yield and compositions of the SLs produced by the gene-deleted mutant of ∆alcs::six showed almost no lactonic form of SLs, and the acidic SLs were composed of medium-chain. The ALCS enzyme was heterologously expressed in Escherichia coli JM109 (DE3) with pMAL-c2x-alcs. The enzyme was purified through a maltose-binding protein (MBP) affinity chromatography column and was confirmed to be homogeneous by SDS-PAGE. The recombinant enzyme could catalyze the formation of the long-chain acyl-CoA when the long-chain fatty acids and the coenzyme A were used as substrates.

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References

  • Abumrad NA, El-Maghrabi MR, Amri EZ, Lopez E, Grimaldi PA (1993) Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation. J Biol Chem 268(24):17665–17668

    CAS  PubMed  Google Scholar 

  • Ashby RD, Solaiman DK, Foglia TA (2006) The use of fatty acid esters to enhance free acid sophorolipid synthesis. Biotechnol Lett 28(4):253–260. doi:10.1007/s10529-005-5527-y

    Article  CAS  PubMed  Google Scholar 

  • Asmer H-J, Lang S, Wagner F, Wray V (1988) Microbial production, structure elucidation and bioconversion of sophorose lipids. J Am Oil Chem Soc 65(9):1460–1466. doi:10.1007/BF02898308

    Article  CAS  Google Scholar 

  • Butler G, Rasmussen MD, Lin MF, Santos MA, Sakthikumar S, Munro CA, Rheinbay E, Grabherr M, Forche A, Reedy JL, Agrafioti I, Arnaud MB, Bates S, Brown AJ, Brunke S, Costanzo MC, Fitzpatrick DA, de Groot PW, Harris D, Hoyer LL, Hube B, Klis FM, Kodira C, Lennard N, Logue ME, Martin R, Neiman AM, Nikolaou E, Quail MA, Quinn J, Santos MC, Schmitzberger FF, Sherlock G, Shah P, Silverstein K, Skrzypek MS, Soll D, Staggs R, Stansfield I, Stumpf MP, Sudbery PE, Srikantha T, Zeng Q, Berman J, Berriman M, Heitman J, Gow NA, Lorenz MC, Birren BW, Kellis M, Cuomo CA (2009) Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature 459(7247):657–662. doi:10.1038/nature08064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ciesielska K, Van Bogaert IN, Chevineau S, Li B, Groeneboer S, Soetaert W, Van de Peer Y, Devreese B (2014) Exoproteome analysis of Starmerella bombicola results in the discovery of an esterase required for lactonization of sophorolipids. J Proteome 98:159–174. doi:10.1016/j.jprot.2013.12.026

    Article  CAS  Google Scholar 

  • Coe NR, Smith AJ, Frohnert BI, Watkins PA, Bernlohr DA (1999) The fatty acid transport protein (FATP1) is a very long-chain acyl-CoA synthetase. J Biol Chem 274(51):36300–36304. doi:10.1074/jbc.274.51.36300

    Article  CAS  PubMed  Google Scholar 

  • Dujon B, Sherman D, Fischer G, Durrens P, Casaregola S, Lafontaine I, De Montigny J, Marck C, Neuvéglise C, Talla E, Goffard N, Frangeul L, Aigle M, Anthouard V, Babour A, Barbe V, Barnay S, Blanchin S, Beckerich JM, Beyne E, Bleykasten C, Boisramé A, Boyer J, Cattolico L, Confanioleri F, De Daruvar A, Despons L, Fabre E, Fairhead C, Ferry-Dumazet H, Groppi A, Hantraye F, Hennequin C, Jauniaux N, Joyet P, Kachouri R, Kerrest A, Koszul R, Lemaire M, Lesur I, Ma L, Muller H, Nicaud JM, Nikolski M, Oztas S, Ozier-Kalogeropoulos O, Pellenz S, Potier S, Richard GF, Straub ML, Suleau A, Swennen D, Tekaia F, Wésolowski-Louvel M, Westhof E, Wirth B, Zeniou-Meyer M, Zivanovic I, Bolotin-Fukuhara M, Thierry A, Bouchier C, Caudron B, Scarpelli C, Gaillardin C, Weissenbach J, Wincker P, Souciet JL (2004) Genome evolution in yeasts. Nature 430:35–44. doi:10.1038/nature02579

    Article  PubMed  Google Scholar 

  • Faergeman NJ, DiRusso CC, Elberger A, Knudsen J, Black PN (1997) Disruption of the Saccharomyces cerevisiae homologue to the murine fatty acid transport protein impairs uptake and growth on long-chain fatty acids. J Biol Chem 272(13):8531–8538. doi:10.1074/jbc.272.13.8531

    Article  CAS  PubMed  Google Scholar 

  • Gorin PAJ, Spencer JFT, Tulloch AP (1961) Hydroxy fatty acid glycosides of sophorose from Torulopsis magnolia. Can J Chem 39(4):846–855. doi:10.1139/v61-104

    Article  CAS  Google Scholar 

  • Hall AM, Wiczer BM, Herrmann T, Stremmel W, Bernlohr DA (2005) Enzymatic properties of purified murine fatty acid transport protein 4 and analysis of acyl-COA synthetase activities in tissues from FATP4 null mice. J Biol Chem 280(12):11848–11954. doi:10.1074/jbc.M412629200

    Article  Google Scholar 

  • Hartmann T, Dumig M, Jaber BM, Szewczyk E, Olbermann P, Morschhäuser J, Krappmann S (2010) Validation of a self-excising marker in the human pathogen Aspergillus fumigatus by employing the β-rec/six site-specific recombination system. Appl Environ Microbiol 76(18):6313–6317. doi:10.1128/AEM.00882-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hu Y, Ju L-K (2001) Purification of lactonic sophorolipids by crystallization. J Biotechnol 87(3):263–272. doi:10.1016/s0168-1656(01)00248-6

    Article  CAS  PubMed  Google Scholar 

  • Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4(3):363–371. doi:10.1038/nprot.2009.2

    Article  CAS  PubMed  Google Scholar 

  • Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845–858. doi:10.1038/nprot.2015.053

    Article  CAS  PubMed  Google Scholar 

  • Kurtzman CP, Price NPJ, Ray KJ, Kuo T-M (2010) Production of sophorolipid biosurfactants by multiple species of the Starmerella (Candida) bombicola yeast clade. FEMS Microbiol Lett 311(2):140–146. doi:10.1111/j.1574-6968.2010.02082.x

    Article  CAS  PubMed  Google Scholar 

  • Li J, Li H, Li W, Xia C, Song X (2016) Identification and characterization of a flavin-containing monooxygenase MoA and its function in a specific sophorolipid molecule metabolism in Starmerella bombicola. Appl Microbiol Biotechnol 100(3):1307–1318. doi:10.1007/s00253-015-7091-2

    Article  CAS  PubMed  Google Scholar 

  • Ma X-J, Li H, Shao L-J, Shen J, Song X (2011) Effects of nitrogen sources on production and composition of sophorolipids by Wickerhamiella domercqiae var. sophorolipid CGMCC 1576. Appl Microbiol Biotechnol 91(6):1623–1632. doi:10.1007/s00253-011-3327-y

    Article  CAS  PubMed  Google Scholar 

  • Ma XJ, Li H, Song X (2012) Surface and biological activity of sophorolipid molecules produced by Wickerhamiella domercqiae var. sophorolipid CGMCC 1576. J Colloid Interface Sci 376(1):165–172. doi:10.1016/j.jcis.2012.03.007

    Article  CAS  PubMed  Google Scholar 

  • Mach RL, Schindler M, Kubicek CP (1994) Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals. Curr Genet 25(6):567–570. doi:10.1007/BF00351679

    Article  CAS  PubMed  Google Scholar 

  • Nakayashiki H, Hanada S, Nguyen BQ, Kadotani N, Tosa Y, Mayama S (2005) RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet Biol 42(4):275–283. doi:10.1016/j.fgb.2005.01.002

    Article  CAS  PubMed  Google Scholar 

  • Pekin G, Vardar-Sukan F, Kosaric N (2005) Production of sophorolipids from Candida bombicola ATCC 22214 using Turkish corn oil and honey. Eng Life Sci 5(4):357–362. doi:10.1002/elsc.200520086

    Article  CAS  Google Scholar 

  • Saerens KMJ, Roelants SLKW, Van Bogaert INA, Soetaert W (2011a) Identification of the UDP-glucosyltransferase gene UGTA1, responsible for the first glucosylation step in the sophorolipid biosynthetic pathway of Candida bombicola ATCC 22214. FEMS Yeast Res 11(1):123–132. doi:10.1111/j.1567-1364.2010.00695.x

    Article  CAS  PubMed  Google Scholar 

  • Saerens KMJ, Zhang J-X, Saey L, Van Bogaert INA, Soetaert W (2011b) Cloning and functional characterization of the UDP-glucosyltransferase UgtB1 involved in sophorolipid production by Candida bombicola and creation of a glucolipid-producing yeast strain. Yeast 28(4):279–292. doi:10.1002/yea.1838

    Article  CAS  PubMed  Google Scholar 

  • Saerens KM, Saey L, Soetaert W (2011c) One-step production of unacetylated sophorolipids by an acetyltransferase negative Candida bombicola. Biotechnol Bioeng 108(12):2923–2931. doi:10.1002/bit.23248

    Article  CAS  PubMed  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425

    CAS  PubMed  Google Scholar 

  • Schaffer JE, Lodish HF (1994) Expression cloning and characterization of a novel adipocyte long chain fatty acid transport protein. Cell 79(3):427–436. doi:10.1016/0092-8674(94)90252-6

    Article  CAS  PubMed  Google Scholar 

  • Servert P, Diaz V, Lucas D, de la Cueva T, Rodriguez M, Garcia-Castro J, Alonso J, Martinez AC, Gonzalez M, Bernad A (2008) In vivo site-specific recombination using the β-rec/six system. Biotechniques 45(1):69–78. doi:10.2144/000112826

    Article  CAS  PubMed  Google Scholar 

  • Spencer JFT, Gorin PA, Tulloch AP (1970) Torulopsis bombicola sp.n. Antonie Van Leeuwenhoek 36(1):129–133. doi:10.1007/BF02069014

    Article  CAS  PubMed  Google Scholar 

  • Stump DD, Zhou SL, Berk PD (1993) Comparison of plasma membrane FABP and mitochondrial isoform of aspartate aminotransferase from rat liver. Am J Phys 265(5):G894–G902

    CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1599. doi:10.1093/molbev/msm092

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739. doi:10.1093/molbev/msr121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thaniyavarn J, Chianguthai T, Sangvanich P, Roongsawang N, Washio K, Morikawa M, Thaniyavarn S (2008) Production of sophorolipid biosurfactant by Pichia anomala. Biosci Biotechnol Biochem 72(8):2061–2068. doi:10.1271/bbb.80166

    Article  CAS  PubMed  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24):4876–4882. doi:10.1093/nar/25.24.4876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tulloch AP, Spencer JFT, Deinema MH (1968) A new hydroxyl fatty acid sophoroside from Candida bogoriensis. Can J Chem 46(3):345–348. doi:10.1139/v68-057

    Article  CAS  Google Scholar 

  • Van Bogaert INA, Saerens K, De Muynck C, Develter D, Soetaert W, Vandamme EJ (2007) Microbial production and application of sophorolipids. Appl Microbiol Biotechnol 76(1):23–34. doi:10.1007/s00253-007-0988-7

    Article  PubMed  Google Scholar 

  • Van Bogaert INA, De Maeseneire SL, Develter D, Soetaert W, Vandamme EJ (2008a) Cloning and characterisation of the glyceraldehyde 3-phosphate dehydrogenase gene of Candida bombicola and use of its promoter. J Ind Microbiol Biotechnol 35(10):1085–1092. doi:10.1007/s10295-008-0386-x

    Article  PubMed  Google Scholar 

  • Van Bogaert INA, De Maeseneire SL, Develter D, Soetaert W, Vandamme EJ (2008b) Development of a transformation and selection system for the glycolipid-producing yeast Candida bombicola. Yeast 25:273–278. doi:10.1002/yea.1586

    Article  PubMed  Google Scholar 

  • Van Bogaert INA, Demey M, Develter D, Soetaert W, Vandamme EJ (2009) Importance of the cytochrome P450 monooxygenase CYP52 family for the sophorolipid-producing yeast Candida bombicola. FEMS Yeast Res 9(1):87–94. doi:10.1111/j.1567-1364.2008.00454.x

    Article  PubMed  Google Scholar 

  • Van Bogaert INA, Zhang J, Soetaert W (2011) Microbial synthesis of sophorolipids. Process Biochem 46(4):821–833. doi:10.1016/j.procbio.2011.01.010

    Article  Google Scholar 

  • Van Bogaert INA, Holvoet K, Roelants SLKW, Li B, Lin Y-C, Van de Peer Y, Soetaert W (2013) The biosynthetic gene cluster for sophorolipids: a biotechnological interesting biosurfactant produced by Starmerella bombicola. Mol Microbiol 88(3):501–509. doi:10.1111/mmi.12200

    Article  PubMed  Google Scholar 

  • Watkins PA (1997) Fatty acid activation. Prog Lipid Res 36:55–83

    Article  CAS  PubMed  Google Scholar 

  • Watkins PA, Lu J-F, Steinberg SJ, Gould SJ, Smith KD, Braiterman LT (1998) Disruption of the Saccharomyces cerevisiae FAT1 gene decreases very long-chain fatty acyl-CoA synthetase activity and elevates intracellular very long-chain fatty acid concentrations. J Biol Chem 273(29):18210–18219. doi:10.1074/jbc.273.29.18210

    Article  CAS  PubMed  Google Scholar 

  • Wodarczak S, Buschmann N (1995) Analytical methods for alkylpolyglucosides. GIT Lab Fachz 5:410–411

    Google Scholar 

  • Yolov AA, Shabarova ZA (1990) Constructing DNA by polymerase recombination. Nucleic Acids Res 18(13):3983–3986. doi:10.1093/nar/18.13.3983

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This study was funded by National Natural Science Foundation of China (No. 30970052 and No. 31270089), Natural Science Foundation of Shandong Province (ZR2009BZ002), and National Key Technology R&D Program (2011BAC02B04).

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  1. State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Shan Da Nan Road 27, Jinan, Shandong, 250100, People’s Republic of China

    Jiashan Li, Chengqiang Xia, Xiaoran Fang, Haizhao Xue & Xin Song

  2. National Glycoengineering Research Center, Shandong University, Shan Da Nan Road 27, Jinan, Shandong, 250100, People’s Republic of China

    Xin Song

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  1. Jiashan Li
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  2. Chengqiang Xia
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Correspondence to Xin Song.

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This article does not contain any studies with human participants or animals performed by any of the authors. The principles of ethical and professional conduct have been followed by all the authors in this study.

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Li, J., Xia, C., Fang, X. et al. Identification and characterization of a long-chain fatty acid transporter in the sophorolipid-producing strain Starmerella bombicola . Appl Microbiol Biotechnol 100, 7137–7150 (2016). https://doi.org/10.1007/s00253-016-7580-y

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