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United States Department of Agriculture

Agricultural Research Service

Research Project: INDUSTRIALLY ROBUST ENZYMES AND MICROORGANISMS FOR PRODUCTION OF SUGARS AND ETHANOL FROM AGRICULTURAL BIOMASS Title: Plasmids for Expression of Heterologous Proteins in Rhizopus Oryzae

Authors
item Mertens, Jeffrey
item Skory, Christopher
item Ibrahim, Ashraf - UCLA MEDICAL CENTER

Submitted to: Archives Of Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 2, 2006
Publication Date: June 1, 2006
Citation: Mertens, J.A., Skory, C.D., Ibrahim, A.S. 2006. Plasmids for expression of heterologous proteins in Rhizopus oryzae. Archives of Microbiology. 186:41-50.

Interpretive Summary: Enzymes are proteins that carry out very specific chemical conversions and are used in numerous applications such as food/beverage preparation, textiles, drug development, and production of fuel ethanol from agricultural crops. Most enzymes used for industrial applications are obtained from microorganisms. There is increasing need for more specialized enzymes in industrial processes; however, many of these enzymes are difficult to produce from organisms that can be used in large scale processes in a safe and cost effective manner. Considerable biotechnology efforts have been made to modify organisms currently used in industry to produce these necessary proteins in large quantities. The fungus Rhizopus is one such organism that is currently used to produce many different enzymes in a safe and cost effective manner. We have now developed technology to allow additional enzymes to be produced in this fungus. This technology is expected to provide a foundation for improved synthesis of numerous enzymes that will benefit a wide variety of industrial processes.

Technical 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 pdc 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.

Last Modified: 4/24/2014
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