A leaf-based assay using interchangeable design principles to rapidly assemble multistep recombinant pathways.
Plant Biotechnol J
; 7(9): 914-24, 2009 Dec.
Article
en En
| MEDLINE
| ID: mdl-19843252
ABSTRACT
The assembly of multistep recombinant pathways in stably transformed plants is a cornerstone of crops producing new products yet can be a laborious and time-consuming process. Any heterologous expression platform capable of providing a rapid estimation of the functional assembly of an entire pathway would guide the design of such transgenic traits. In this study, we use a Nicotiana benthamiana transient leaf expression system to simultaneously express five genes, from five independent T(DNA) binary vectors, to assemble a complete recombinant pathway in five days. In this study, we demonstrate the production of long-chain polyunsaturated fatty acids (LC-PUFA) requiring five transgene-encoded reactions to convert endogenous fatty acids to LC-PUFA. The addition of a triacylglycerol assembly enzyme, Arabidopsis thaliana diacylglyceride-O-acyltransferase, and fractionation of the total lipid profile demonstrated that leaf oils contained 37% newly synthesised LC-PUFA, including 7% arachidonic acid (AA), 6% eicosopentaenoic acid and 3% docosahexaenoic acid. The calculation of enzymatic conversion efficiencies at each step of LC-PUFA synthesis suggests that this transient assembly of a complicated multistep pathway is highly efficient. Unlike experiments using stably transformed plants our assembly of an intricate pathway maintained full gene-for-gene interchangeability and required a fraction of the time and glasshouse space. Furthermore, an exogenous LC-PUFA fatty acid substrate, AA, was fed and metabolised by a transiently expressed Delta17-desaturase enzyme, and provided results similar to those obtained in yeast feeding experiments. Although the assay was ideal for LC-PUFA pathways, this assay format may become a powerful tool for the characterisation and step-wise improvement of other recombinant pathways and multigenic traits.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Nicotiana
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Ácidos Grasos Insaturados
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Vectores Genéticos
Idioma:
En
Revista:
Plant Biotechnol J
Asunto de la revista:
BIOTECNOLOGIA
/
BOTANICA
Año:
2009
Tipo del documento:
Article
País de afiliación:
Australia