transient-gene-expression-tobacco-using-gibson-assembly-gene

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Harvard University

Harvard Medical School

The 3-hydroxypropionate (3-HPA) bicycle is unique among CO2-fixing systems in that none of its enzymes appear to be affected by oxygen. Moreover, the bicycle includes a number of enzymes that produce novel intermediates of biotechnological interest, and the CO2-fixing steps in this pathway are relatively rapid. We expressed portions of the 3-HPA bicycle in a heterologous organism, E. coli K12. We subdivided the 3-HPA bicycle into four sub-pathways: (1) synthesis of propionyl-CoA from acetyl-CoA, (2) synthesis of succinate from propionyl-CoA, (3) glyoxylate production and regeneration of acetyl-CoA, and (4) assimilation of glyoxylate and propionyl-CoA to form pyruvate and regenerate acetyl-CoA. We expressed the novel enzymes of the 3-HPA bicycle in operon form and used phenotypic tests for activity. Sub-pathway 1 activated a propionate-specific biosensor. Sub-pathway 2, found in non-CO2-fixing bacteria, was reassembled in E. coli using genes from diverse sources. Sub-pathway 3, operating in reverse, generated succinyl-CoA sufficient to rescue a sucAD(-) double mutant of its diaminopimelic acid (DAP) auxotrophy. Sub-pathway 4 was able to reduce the toxicity of propionate and allow propionate to contribute to cell biomass in a prpC(-)(2 methylcitrate synthase) mutant strain. These results indicate that all of the sub-pathways of the 3-HPA bicycle can function to some extent in vivo in a heterologous organism, as indicated by growth tests. Overexpression of certain enzymes was deleterious to cell growth, and, in particular, expression of MMC-CoA lyase caused a mucoid phenotype. These results have implications for metabolic engineering and for bacterial evolution through horizontal gene transfer.

Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth.

Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments.

Article	Year
Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth. Metabolic engineering\| PubMed ID: 23376595	2013
Targeting a heterologous protein to multiple plant organelles via rationally designed 5' mRNA tags. Journal of biological engineering\| PubMed ID: 24011257	2013

Matthew D. Mattozzi

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