Genetic engineering of a Lemna isoleucine auxotroph

Lemna, a member of the Lemnaceae or duckweed family, is a small aquatic plant that can be quickly transformed to produce recombinant proteins in a contained and controlled bioprocessing environment. The containment capability of Lemna has been further improved with the creation of an auxotroph platform that requires isoleucine supplementation for survival of transformed plant lines. Using an RNAi based approach, threonine deaminase (TD) expression was targeted and thus resulted in dramatically reduced expression of this key enzyme in the isoleucine biosynthesis pathway. Auxotrophic plants expressing RNAi for TD were generated in the presence of isoleucine and selected based on their inability to propagate without isoleucine supplementation. TD transcripts isolated from the superior auxotroph lines were shown to be less than 10% of wild type level and thus confirmed the auxotroph phenotype to be derived from the specific knock down of TD expression. When grown under optimal conditions with appropriate isoleucine supplementation, biomass accumulation of the auxotroph lines was equivalent to that of wild type plants. To demonstrate the application of this system for production of recombinant proteins, an avian influenza H5N1 hemagglutinin (HA) protein was expressed in the isoleucine auxotroph platform. The successful expression of H5N1 HA vaccine antigen, in the isoleucine auxotroph background demonstrates the applicability of using an auxotroph to express biotherapeutics and vaccines in a highly contained expression system.