A leaf-based assay using interchangeable design principles to rapidly assemble multistep recombinant pathways

Plant Biotechnol J. 2009 Dec;7(9):914-24. doi: 10.1111/j.1467-7652.2009.00453.x. Epub 2009 Oct 13.


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.

MeSH terms

  • Acetyltransferases / genetics
  • Acetyltransferases / metabolism
  • Fatty Acid Elongases
  • Fatty Acids, Unsaturated / biosynthesis*
  • Gene Expression Regulation, Plant
  • Genes, Plant
  • Genetic Vectors*
  • Plant Leaves / genetics
  • Plant Leaves / metabolism
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / metabolism
  • Plasmids
  • Rhizobium
  • Tobacco / genetics
  • Tobacco / metabolism*
  • Transgenes


  • Fatty Acids, Unsaturated
  • Acetyltransferases
  • Fatty Acid Elongases