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.
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We thank Ryan Burdick and Kristina Glenzinski for their technical expertise in completing this work and Michael Cotta for critical reading of the manuscript. ASI is supported by National Institute of Health grant R56 AI063503-01A1.
USDA Mention of trade names or commercial products used in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.
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Mertens, J.A., Skory, C.D. & Ibrahim, A.S. Plasmids for expression of heterologous proteins in Rhizopus oryzae . Arch Microbiol 186, 41–50 (2006). https://doi.org/10.1007/s00203-006-0121-9
- Rhizopus oryzae
- Green fluorescent protein
- Heterologous protein expression
- Gene copy number