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Selection and characterization of promoters based on genomic approach for the molecular breeding of oleaginous fungus Mortierella alpina 1S-4

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Abstract

To express a foreign gene effectively, a good expression system is required. In this study, we investigated various promoters as useful tools for gene manipulation in oleaginous fungus Mortierella alpina 1S-4. We selected and cloned the promoter regions of 28 genes in M. alpina 1S-4 on the basis of expression sequence tag abundance data. The activity of each promoter was evaluated using the β-glucuronidase (GUS) reporter gene. Eight of these promoters were shown to enhance GUS expression more efficiently than a histone promoter, which is conventionally used for the gene manipulation in M. alpina. Especially, the predicted protein 3 and the predicted protein 6 promoters demonstrated approximately fivefold higher activity than the histone promoter. The activity of some promoters changed along with the cultivation phase of M. alpina 1S-4. Seven promoters with constitutive or time-dependent, high-level expression activity were selected, and deletion analysis was carried out to determine the promoter regions required to retain activity. This is the first report of comprehensive promoter analysis based on a genomic approach for M. alpina. The promoters described here will be useful tools for gene manipulation in this strain.

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References

  • Ando A, Sakuradani E, Horinaka K, Ogawa J, Shimizu S (2009a) Transformation of an oleaginous zygomycete Mortierella alpina 1S-4 with the carboxin resistance gene conferred by mutation of the iron-sulfur subunit of succinate dehydrogenase. Curr Genet 55:349–356

    Article  CAS  PubMed  Google Scholar 

  • Ando A, Sumida Y, Negoro H, Suroto DA, Ogawa J, Sakuradani E, Shimizu S (2009b) Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous fungus, Mortierella alpina 1S-4, and its application for eicosapentaenoic acid producer breeding. Appl Environ Microbiol 75:5529–5535

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Archer DB, Jeenes DJ, Mackenzie DA (1994) Strategies for improving heterologous protein production from filamentous fungi. Antonie Van Leeuwenhoek 65:245–250

    Article  CAS  PubMed  Google Scholar 

  • Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421

    Article  CAS  PubMed  Google Scholar 

  • Beopoulos A, Nicaud JM, Gaillardin C (2011) An overview of lipid metabolism in yeasts and its impact on biotechnological processes. Appl Microbiol Biotechnol 90:1193–1206

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66

    Article  CAS  PubMed  Google Scholar 

  • Courchesne NMD, Parisien A, Wang B, Lan CQ (2009) Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J Biotechnol 141:31–41

    Article  CAS  PubMed  Google Scholar 

  • Gill I, Valivety R (1997) Polyunsaturated fatty acids, part 1: occurrence, biological activities and applications. Trends Biotechnol 15:401–409

    Article  CAS  PubMed  Google Scholar 

  • Hansson L, Blackberg L, Edlund M, Lundberg L, Stromqvist M, Hernell O (1993) Recombinant human milk bile salt-stimulated lipase. Catalytic activity is retained in the absence of glycosylation and the unique proline-rich repeats. J Biol Chem 268:26692–26698

    CAS  PubMed  Google Scholar 

  • Hata Y, Tsuchiya K, Kitamoto K, Gomi K, Kumagai C, Tamura G, Hara S (1991) Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae. Gene 108:145–150

    Article  CAS  PubMed  Google Scholar 

  • Ichishima E, Taya N, Ikeguchi M, Chiba Y, Nakamura M, Kawabata C, Inoue T, Takahashi K, Minetoki T, Ozeki K, Kumagai C, Gomi K, Yoshida T, Nakajima T (1999) Molecular and enzymic properties of recombinant 1,2-α-mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells. Biochem J 339:589–597

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jareonkitmongkol S, Kawashima H, Shirasaka N, Shimizu S, Yamada H (1992) Production of dihomo-γ-linolenic acid by a Δ5-desaturase-defective mutant of Mortierella alpina 1S-4. Appl Environ Microbiol 58:2196–2200

    CAS  PubMed Central  PubMed  Google Scholar 

  • Jareonkitmongkol S, Shimizu S, Yamada H (1993) Production of an eicosapentaenoic acid-containing oil by a Δ12 desaturase-defective mutant of Mortierella alpina 1S-4. J Am Oil Chem Soc 70:119–123

    Article  CAS  Google Scholar 

  • Jefferson RA, Burgess SM, Hirsh D (1986) β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci 83:8447–8451

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed Central  PubMed  Google Scholar 

  • Kawashima H, Kamada N, Sakuradani E, Jareonkitmongkol S, Akimoto K, Shimizu S (1997) Production of 8,11,14,17-cis-eicosatetraenoic acid by Δ5 desaturase-defective mutants of an arachidonic acid-producing fungus, Mortierella alpina. J Am Oil Chem Soc 74:455–459

    Article  CAS  Google Scholar 

  • Kusakabe T, Koga K, Sugimoto Y (1994) Isolation and characterization of cDNA and genomic promoter region for a heat shock protein 30 from Aspergillus nidulans. Biochim Biophys Acta Gene Struct Expr 1219:555–558

    Article  CAS  Google Scholar 

  • MacKenzie DA, Jeenes DJ, Belshaw NJ, Archer DB (1993) Regulation of secreted protein production by filamentous fungi: recent developments and perspectives. J Gen Microbiol 139:2295–2307

    Article  CAS  PubMed  Google Scholar 

  • Mackenzie DA, Wongwathanarat P, Carter AT, Archer DB (2000) Isolation and use of a homologous histone H4 promoter and a ribosomal DNA region in a transformation vector for the oil-producing fungus Mortierella alpina. Appl Environ Microbiol 66:4655–4661

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538

    CAS  PubMed Central  PubMed  Google Scholar 

  • Minetoki T, Kumagai C, Gomi K, Kitamoto K, Takahashi K (1998) Improvement of promoter activity by the introduction of multiple copies of the conserved region III sequence, involved in the efficient expression of Aspergillus oryzae amylase-encoding genes. Appl Microbiol Biotechnol 50:459–467

    Article  CAS  PubMed  Google Scholar 

  • Mumberg D, Muller R, Funk M (1994) Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res 22:5767–5768

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ostergaard S, Olsson L, Nielsen J (2000) Metabolic engineering of Saccharomyces cerevisiae. Microbiol Mol Biol Rev 64:34–50

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Punt PJ, Van Biezen N, Conesa A, Albers A, Mangnus J, Van Den Hondel C (2002) Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol 20:200–206

    Article  CAS  PubMed  Google Scholar 

  • Raghukumar S (2008) Thraustochytrid marine protists: production of PUFAs and other emerging technologies. Mar Biotechnol 10:631–640

    Article  CAS  PubMed  Google Scholar 

  • Ranamalie Amarasinghe BHR, Faivre-Nitschke E, Wu Y, Udall JA, Dennis ES, Constable G, Llewellyn DJ (2006) Genomic approaches to the discovery of promoters for sustained expression in cotton (Gossypium hirsutum L.) under field conditions: expression analysis in transgenic cotton and Arabidopsis of a Rubisco small subunit promoter identified using EST sequence analysis and cDNA microarrays. Plant Biotechnol J 23:437–450

    Article  Google Scholar 

  • Ratledge C (1993) Single cell oils —have they a biotechnological future? Trends Biotechnol 11:278–284

    Article  CAS  PubMed  Google Scholar 

  • Sakuradani E, Kobayashi M, Shimizu S (1999) Δ9-Fatty acid desaturase from arachidonic acid-producing fungus. Unique gene sequence and its heterologous expression in a fungus, Aspergillus. Eur J Biochem 260:208–216

    Article  CAS  PubMed  Google Scholar 

  • Sakuradani E, Ando A, Shimizu S, Ogawa J (2013) Metabolic engineering for the production of polyunsaturated fatty acids by oleaginous fungus Mortierella alpina 1S-4. J Biosci Bioeng 116:417–422

    Article  CAS  PubMed  Google Scholar 

  • Shen WJ, Forde BG (1989) Efficient transformation of Agrobacterium spp. by high voltage electroporation. Nucleic Acids Res 17:8385

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Shimizu S, Ogawa J, Kataoka M, Kobayashi M (1997) Screening of novel microbial enzymes for the production of biologically and chemically useful compounds. Adv Biochem Eng 58:45–87

    CAS  Google Scholar 

  • Sumi A, Okuyama K, Kobayashi K, Ohtani W, Ohmura T, Yokoyama K (1999) Purification of recombinant human serum albumin efficient purification using STREAMLINE. Bioseparation 8:195–200

    Article  CAS  PubMed  Google Scholar 

  • Tada S, Gomi K, Kitamoto K, Takahashi K, Tamura G, Hara S (1991) Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae. Mol Gen Genet 229:301–306

    Article  CAS  PubMed  Google Scholar 

  • Takeno S, Sakuradani E, Murata S, Inohara-Ochiai M, Kawashima H, Ashikari T, Shimizu S (2004a) Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4. Biosci Biotechnol Biochem 68:277–285

    Article  CAS  PubMed  Google Scholar 

  • Takeno S, Sakuradani E, Murata S, Inohara-Ochiai M, Kawashima H, Ashikari T, Shimizu S (2004b) Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4. Appl Microbiol Biotechnol 65:419–425

    Article  CAS  PubMed  Google Scholar 

  • Takeno S, Sakuradani E, Tomi A, Inohara-Ochiai M, Kawashima H, Shimizu S (2005) Transformation of oil-producing fungus, Mortierella alpina 1S-4, using Zeocin, and application to arachidonic acid production. J Biosci Bioeng 100:617–622

    Article  CAS  PubMed  Google Scholar 

  • Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R (2003) Molecular farming in plants: host systems and expression technology. Trends Biotechnol 21:570–578

    Article  CAS  PubMed  Google Scholar 

  • Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was partially supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) to J.O.; Advanced Low Carbon Technology Research and Development Program (ALCA) to J.S.; Grants-in-Aid for Scientific Researches (no. 22380051) from the Ministry of Education, Science, Sports, and Culture of Japan to E.S.; and Institute for Fermentation, Osaka (IFO).

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Authors and Affiliations

  1. Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8052, Japan

    Tomoyo Okuda, Akinori Ando, Eiji Sakuradani, Hiroshi Kikukawa & Jun Ogawa

  2. Research Unit for Physiological Chemistry, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8052, Japan

    Akinori Ando

  3. Research Institute, Suntory Global Innovation Center Ltd., 1-1-1 Wakayamadai Shimamoto-cho, Mishima-gun, Osaka, 618-8503, Japan

    Nozomu Kamada & Misa Ochiai

  4. Research Division of Microbial Sciences Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8052, Japan

    Jun Shima

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  1. Tomoyo Okuda
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  2. Akinori Ando
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  3. Eiji Sakuradani
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  4. Hiroshi Kikukawa
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  6. Misa Ochiai
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  7. Jun Shima
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  8. Jun Ogawa
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Correspondence to Jun Ogawa.

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Communicated by Stefan Hohmann.

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Okuda, T., Ando, A., Sakuradani, E. et al. Selection and characterization of promoters based on genomic approach for the molecular breeding of oleaginous fungus Mortierella alpina 1S-4. Curr Genet 60, 183–191 (2014). https://doi.org/10.1007/s00294-014-0423-4

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  • DOI: https://doi.org/10.1007/s00294-014-0423-4

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