A leaf-based assay using interchangeable design principles to rapidly assemble multistep recombinant pathways.
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.
Wood
Craig C
CC
CSIRO Plant Industry, Canberra, ACT, Australia. craig.wood@csiro.au
Petrie
James R
JR
Shrestha
Pushkar
P
Mansour
Maged P
MP
Nichols
Peter D
PD
Green
Allan G
AG
Singh
Surinder P
SP
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D000081031:Fatty Acid Elongases; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005822:Genetic Vectors; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D012231:Rhizobium; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes
D005231:Fatty Acids, Unsaturated; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases