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Role of N-terminal 28-amino-acid region of Rhizopus oryzae lipase in directing proteins to secretory pathway of Aspergillus oryzae

Abstract

To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL–green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL–GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.

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Acknowledgement

This work was cofinanced by the Research and Development Program for New Bio-industry Initiatives and by New Energy and Industrial Technology Development Organization (NEDO), Japan. We are grateful to Dr. Toshitaka Minetoki (Ozeki Co., Hyogo, Japan) who kindly provided the A. oryzae niaD300 strain, pNGA142, and pNAN8142. We also thank Dr. Masaru Kaieda (Canon Inc., Tokyo, Japan) for invaluable technical advice when he was at Kobe University.

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Correspondence to Akihiko Kondo.

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Hama, S., Tamalampudi, S., Shindo, N. et al. Role of N-terminal 28-amino-acid region of Rhizopus oryzae lipase in directing proteins to secretory pathway of Aspergillus oryzae . Appl Microbiol Biotechnol 79, 1009 (2008). https://doi.org/10.1007/s00253-008-1502-6

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Keywords

  • Filamentous fungi
  • Protein secretion
  • Heterologous protein production
  • N-terminal peptide
  • Green fluorescent protein
  • Visualization