Mutiple gene expression for engineering novel pathways and hyperexpression of foreign proteins in plants

Abstract

Introducing blocks of foreign genes in a single operon would avoid complications such as position effect and gene silencing inherent in putting one gene at a time into random locations in the nuclear genome. Cloning several genes into a single T-DNA does not avoid the compounded variable expression problem encountered in nuclear transgenic plants. This disclosure shows that a bacterial operon can be expressed in a single integration event as opposed to multiple events requiring several years to accomplish. Expression of multiple genes via a single transformation event opens the possibility of expressing foreign pathways or pharmaceutical proteins involving multiple genes. Expressing the Cry2aA2 operon, including a putative chaperonin to aid in protein folding, in the chloroplast via a single transformation event leads to production of crystalized insecticidal proteins. Expressing the Mer operon via a single transformation event leads to a phytoremediation system.

Description

[0001] This patent application claims the benefit of U.S. Provisional Applications Nos. 60 / 185,660, filed Feb. 29, 2000, 60 / 257,408, filed Dec. 22, 2000, 60 / 259,248 filed Dec. 29, 2000 and 60 / 266,121 filed Feb. 2, 2001. All applications are here incorporated by reference.[0003] This application pertains to the field of genetic engineering of plant genomes, particularly plastids and to methods of and engineered plants with operons that lead to and result in overexpression of the gene of interest. This application also pertains to the field of genetic engineering of algal and bacterial genomes.DESCRIPTION OF RELATED ART[0004] Karamata, in U.S. Pat. No. 4,797,279, proposed the generation of Bacillus thuringiensis hybrids that have insecticidal properties through conjugation. Conjugation is mediated by a conjugative plasmid functional in the B.t. kurstaki strain and the B.t. tenebrionis strain. The resulting hybrid is capable of producing each of the delta-endotoxin crystals typical for...

AI Technical Summary

Benefits of technology

[0022] By this invention, plastid expression constructs are provided which are useful for genetic engineering of plant cells and which provide for enhanced expression of several foreign proteins in plastids utilizing a single transformation event. The transformed plastid is preferrably a metabolically active plastid, such as the chloroplasts found in green and non-green plant tissues including leaves and other parts of the plant. This invention opens the door to engineering novel pathways for metabolic engineering and gene stacking, or for multi subunit complex proteins requiring stoichiometric and coordinated expression of multiple genes. The plastid is preferably one which is maintained at a high copy number in the plant tissue of interest.

[0046] The invention also provides an environment friendly approach to engineering insect resistance to plants because folded crystal products improve the safety of the Bt transgenic plants which will be edible consume, mammals, including humans.

Problems solved by technology

This poses a serious problem when engineering multiple genes in plants.

In the past other techniques, such as mechanical and bacterial bioremediation were implemented with little success, since they were costly and threatened the safety of our environment.

Organomercurials are more toxic due to its increased hydrophobicity, which allows it to cross lipid membranes because it is more hydrophobic than mercury.

In water, mercury pollution also poses a problem.

During their life span, they can accumulate high levels of methylmercury that can reach 1.0 ppm in normal water and 30 ppm in areas of high pollution with mercury (http: / / ehpnet.niehs.nih.gov) Then, humans and birds feed on contaminated fish and accumulation in their tissue cause severe neurological damage.

One of the drawbacks of nuclear genetic engineerig is that it requires several back crosses to create the complete pathway that detoxifies mercury and organomercurials (Bizily et al.

Polycistrons have been observed in chloroplasts in the past but processing RNA sequences present in between individual transcripts, proteins or enzymes involved in processing or cofactors necessary for processing of polycistrons have not yet been characterized Therefore, it was not obvious to one skilled in the art that multiple foreign gene transcripts would be properly processed and translated when expressed from a heterologous promoter.

Prior to this patent application there were no published reports of expression of multiple genes in chloroplasts and there were valid reasons to suggest that it would be problematic.

Indeed, despite several reports of foreign gene expression via the chloroplast genome, no one ever attempted expression of a bacterial operon via the chloroplast genome because of inadequate understanding of processing of polycistrons within plastids.

Also, it was not anticipated that a chaperonin present in a bacterial cell would function within chloroplasts and help fold the foreign protein or interfere with folding of other chloroplast proteins.

It was certainly unanticipated that it was possible to create cuboidal crystals within chloroplasts duplicating the functions of a bacterium during sporulation or duplicate bioremediation pathways within plastids.

Indeed, the prior art suggested that there might have been unforeseen deleterious effects of high-level expression of several foreign proteins within chloroplasts on plant growth or development that were not apparent from the experiences with other transgenes.

Engineering multiple genes in transgenic plants via the nuclear genome is not only extremely time consuming (taking several years to accomplish) but is riddled with problems of position effect, gene silencing etc.

Cloning several genes into a single T-DNA does not avoid the compounded variable expression problem encountered in nuclear transgenic plants.

Relying only on currently available biological and chemical processing, the efflux rates of Hg(0) from mercury contaminated sites are extremely slow.

Attempts to completely solubilize crystalline inclusion bodies for SDS PAGE analysis were not successful because higher pH interfered with gel electrophoresis and repeated dilution decreased protein concentration below detectable levels in Coomassie stained gels.

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