Submitted to: Society of Industrial Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 8/25/2005
Publication Date: 8/25/2005
Citation: Mertens, J.A., Skory, C.D. 2005. Development of plasmids for expression of heterologous proteins in Rhizopus oryzae [abstract]. Society of Industrial Microbiology. p. 100.
Technical Abstract: Rhizopus oryzae has long been used for enzyme production (e.g., glucoamylase and lipase), organic acid synthesis, and various fermented food applications. Our laboratory has made significant advances for over-expression of homologous genes by incorporation of multiple copies, either episomally or integrated into the genome. In this work, we describe a set of plasmid-based expression vectors that can be used for the production of heterologous proteins in Rhizopus oryzae. Three plasmid vectors have been created using either the glucoamylase A (amyA), pyruvate decarboxylase (pdc), or phosphoglycerate kinase (pgk) 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. As a proof of concept, we have expressed green fluorescent protein (GFP) and compared transcription and protein production for each of the expression vectors. The pdc and pgk promoters gave the highest and lowest production rates, respectively. The amyA promoter demonstrates very weak expression with glucose, but was induced to levels intermediate to the pdc and pgk promoters when using cellobiose or soluble starch as the carbon source. These vectors should be useful for overexpression of heterologous proteins and potentially, metabolic engineering of Rhizopus strains.