Archives of Microbiology

, Volume 186, Issue 1, pp 41–50

Plasmids for expression of heterologous proteins in Rhizopus oryzae

  • Jeffrey A. Mertens
  • Christopher D. Skory
  • Ashraf S. Ibrahim
Original Paper

DOI: 10.1007/s00203-006-0121-9

Cite this article as:
Mertens, J.A., Skory, C.D. & Ibrahim, A.S. Arch Microbiol (2006) 186: 41. doi:10.1007/s00203-006-0121-9

Abstract

Rhizopus oryzae has long been used for enzyme production (e.g., glucoamylase and lipase), organic acid synthesis, and various fermented food applications. In this work, we describe a set of plasmid-based expression vectors that can be used for the production of heterologous proteins in R. oryzae. Three plasmid vectors have been created using either the glucoamylase A (amyA), pyruvate decarboxylase (pdcA), or phosphoglycerate kinase (pgk1) promoters to drive expression of heterologous proteins. All three plasmids use the pdcA terminator for transcription termination, the pyrG gene for restoration of uracil prototrophy, and an ampicillin resistance gene and origin of replication for maintenance in Escherichia coli. We have expressed green fluorescent protein (GFP) and compared transcription and protein accumulation for each of the expression vectors. Accumulation of GFP transcript and protein was directly correlated with the choice of promoter with pdcA amyA pgk1. Transcript level appears to parallel GFP protein accumulation. Plasmid copy number had little impact on transcription or protein accumulation. These vectors should be useful for overexpression of heterologous proteins and potentially, metabolic engineering of Rhizopus strains.

Keywords

Rhizopus oryzaeGreen fluorescent proteinHeterologous protein expressionGene copy numberRT-PCR

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Jeffrey A. Mertens
    • 1
  • Christopher D. Skory
    • 2
  • Ashraf S. Ibrahim
    • 3
  1. 1.Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDAAgricultural Research ServicePeoriaUSA
  2. 2.Bioproducts and Biocatalysis Research Unit, National Center for Agricultural Utilization Research, USDAAgricultural Research ServicePeoriaUSA
  3. 3.Los Angeles Biomedical Research Institute at HarborUCLA Medical CenterTorranceUSA