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Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae

  • Bioenergy and Biofuels
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Abstract

In the present study, a system with high lipase expression in Aspergillus oryzae was developed using an improved enolase promoter (P-enoA124) and the 5′ untranslated region of a heat-shock protein (Hsp-UTR). P-enoA142 enhanced the transcriptional level of a heterologous lipase gene and Hsp-UTR improved its translational efficiency. Fusarium heterosporum lipase (FHL) was inserted into a pSENSU-FHL expression vector harboring P-enoA142 and Hsp-UTR and was transformed into an A. oryzae NS4 strain. Transformants possessing pSENSU-FHL in single (pSENSU-FHL#1) and double copies (pSENSU-FHL#2) were selected to evaluate the lipase activity of the whole-cell biocatalyst. The two strains, pSENSU-FHL#1 and #2, showed excellent lipase activity in hydrolysis compared with the strain transformed with conventional expression vector pNAN8142-FHL. Furthermore, by using pSENSU-FHL#2, methanolysis could proceed much more effectively without deactivation, which allowed a swift addition of methanol to the reaction mixture, thereby reducing reaction time.

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References

  • Ban K, Kaieda M, Matsumoto T, Kondo A, Fukuda H (2001) Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochem Eng J 8:39–43

    Article  CAS  Google Scholar 

  • Ban K, Hama S, Nishizuka K, Kaieda M, Matsumoto T, Kondo A, Noda H, Fukuda H (2002) Repeated use of whole-cell biocatalysts immobilized within biomass support particles for biodiesel fuel production. J Mol Catal B Enzym 17:157–165

    Article  CAS  Google Scholar 

  • Buxton FP, Cwynne DI, Davies WR (1989) Cloning of a new bidirectionally selectable marker for Aspergillus strains. Gene 84:329–334

    Article  CAS  Google Scholar 

  • Chen JW, Wu WT (2003) Regeneration of immobilized Candida antarctica lipase for transesterification. J Biosci Bioeng 95:466–469

    Article  CAS  Google Scholar 

  • Clark SJ, Wangner L, Schrock MD, Piennaar PG (1984) Methyl and ethyl soybean esters as renewable fuels for diesel engines. J Am Oil Chem Soc 61:1632–1638

    Article  CAS  Google Scholar 

  • Fukuda H, Kondo A, Noda H (2001) Biodiesel fuel production by transesterification of oils. J Biosci Bioeng 92:405–416

    Article  CAS  Google Scholar 

  • Fukuda H, Hama S, Tamalampudi S, Noda H (2008) Whole-cell biocatalysts for biodiesel fuel production. Trends Biotechnol 26:668–673

    Article  CAS  Google Scholar 

  • Gomi K, Iimura Y, Hara S (1987) Integrative transformation of Aspergillus oryzae with a plasmid containing Aspergillus nidulans argB gene. Agric Biol Chem 51:2549–2555

    CAS  Google Scholar 

  • Hama S, Tamalampudi S, Suzuki Y, Yoshida A, Fukuda H, Kondo A (2008) Preparation and comparative characterization of immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase for enzymatic biodiesel production. Appl Microbiol Biotechnol 81:637–645

    Article  CAS  Google Scholar 

  • Hama S, Numata T, Tamalampudi S, Yoshida A, Noda H, Kondo A, Fukuda H (2009) Use of mono- and diacylglycerol lipase as immobilized fungal whole cells to convert residual partial glycerides enzymatically into fatty acid methyl esters. J Mol Catal B Enzym 58:93–97

    Article  CAS  Google Scholar 

  • Kaieda M, Samukawa T, Matsumoto T, Ban K, Kondo A, Shimada Y, Noda H, Nomoto F, Ohtsuka K, Izumoto E, Fukuda H (1999) Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent. J Biosci Bioeng 88:627–631

    Article  CAS  Google Scholar 

  • Koda A, Bogaki T, Minetoki T, Hirotsune M (2006) 5′ Untranslated region of the HSP 12 gene contributes to efficient translation in Aspergillus oryzae. Appl Microbiol Biotechnol 70:333–336

    Article  CAS  Google Scholar 

  • Kozak M (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266:19867–19870

    CAS  PubMed  Google Scholar 

  • Ma F, Hanna MA (1999) Biodiesel production: a review. Bioresour Technol 70:1–15

    Article  CAS  Google Scholar 

  • Machida M, Chang YC, Manabe M, Yasukawa M, Kunihiro S, Jigami Y (1996) Molecular cloning of a cDNA encoding enolase from the filamentous fungus, Aspergillus oryzae. Curr Genet 30:423–431

    Article  CAS  Google Scholar 

  • Morris DR, Geballe AP (2000) Upstream open reading frames as regulators of mRNA translation. Mol Cell Biol 20:8635–8642

    Article  CAS  Google Scholar 

  • Oda M, Kaieda M, Hama S, Yamaji H, Kondo A, Izumoto E, Fukuda H (2005) Facilitatory effect of immobilized lipase-producing Rhizopus oryzae cells on acyl migration in biodiesel-fuel production. Biochem Eng J 23:45–51

    Article  CAS  Google Scholar 

  • Shimada Y, Watanabe Y, Samukawa T, Sugihara A, Noda H, Fukuda H, Tominaga Y (1999) Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase. J Am Oil Chem Soc 76:789–793

    Article  CAS  Google Scholar 

  • Shimada Y, Watanabe Y, Sugihara A, Tominaga Y (2002) Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J Mol Catal B Enzym 17:133–142

    Article  CAS  Google Scholar 

  • Tamalampudi S, Talukder MMR, Hama S, Tanino T, Suzuki Y, Kondo FH (2007) Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica. Appl Microbiol Biotechnol 75:387–395

    Article  CAS  Google Scholar 

  • Tsuboi H, Koda A, Toda T, Minetoki T, Hirotsune M, Machida M (2005) Improvement of the Aspergillus oryzae enolase promoter (P-enoA) by the introduction of cis-element repeats. Biosci Biotechnol Biochem 69(1):206–208

    Article  CAS  Google Scholar 

  • Unkles SE, Campbell EI, de Ruiter-Jacobs YMJT, Broekhuijsen M, Macro JA, Carrez D, Contreras R, van den Hondel CAMJJ, Kinghorn JR (1989) The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway: a convenient and general selection system for filamentous fungal transformation. Mol Gen Genet 218:99–104

    Article  CAS  Google Scholar 

  • Yamada O, Lee BR, Gomi K (1997) Transformation system for Aspergillus oryzae with double auxotrophic mutations, niaD and sC. Biosci Biotechnol Biochem 61(8):1367–1369

    Article  CAS  Google Scholar 

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Acknowledgments

This work was partially supported by Regional Innovation Creation R&D Programs, the Ministry of Economy, Trade and Industry (METI), and Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan.

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Authors and Affiliations

  1. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1–1 Rokkoudaicho, Nada-ku, Kobe, 657–8501, Japan

    Tomohiro Takaya, Risa Koda, Daisuke Adachi, Chiaki Ogino & Akihiko Kondo

  2. Organization of Advanced Science and Technology, Kobe University, Kobe, Japan

    Kazunori Nakashima

  3. Ozeki Co., 4–9 Imazu Dezaike-cho, Nishinomiya-shi, Hyogo, 663–8227, Japan

    Junpei Wada & Takayuki Bogaki

Authors
  1. Tomohiro Takaya
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  2. Risa Koda
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  3. Daisuke Adachi
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  4. Kazunori Nakashima
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  5. Junpei Wada
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  6. Takayuki Bogaki
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  7. Chiaki Ogino
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  8. Akihiko Kondo
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Correspondence to Akihiko Kondo.

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Takaya, T., Koda, R., Adachi, D. et al. Highly efficient biodiesel production by a whole-cell biocatalyst employing a system with high lipase expression in Aspergillus oryzae . Appl Microbiol Biotechnol 90, 1171–1177 (2011). https://doi.org/10.1007/s00253-011-3186-6

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  • DOI: https://doi.org/10.1007/s00253-011-3186-6

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