Document Detail


A leaf-based assay using interchangeable design principles to rapidly assemble multistep recombinant pathways.
MedLine Citation:
PMID:  19843252     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Craig C Wood; James R Petrie; Pushkar Shrestha; Maged P Mansour; Peter D Nichols; Allan G Green; Surinder P Singh
Publication Detail:
Type:  Journal Article     Date:  2009-10-13
Journal Detail:
Title:  Plant biotechnology journal     Volume:  7     ISSN:  1467-7652     ISO Abbreviation:  Plant Biotechnol. J.     Publication Date:  2009 Dec 
Date Detail:
Created Date:  2009-11-12     Completed Date:  2010-01-14     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101201889     Medline TA:  Plant Biotechnol J     Country:  England    
Other Details:
Languages:  eng     Pagination:  914-24     Citation Subset:  IM    
Affiliation:
CSIRO Plant Industry, Canberra, ACT, Australia. craig.wood@csiro.au
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MeSH Terms
Descriptor/Qualifier:
Acetyltransferases / genetics,  metabolism
Fatty Acids, Unsaturated / biosynthesis*
Gene Expression Regulation, Plant
Genes, Plant
Genetic Vectors*
Plant Leaves / genetics,  metabolism
Plants, Genetically Modified / genetics,  metabolism
Plasmids
Rhizobium
Tobacco / genetics,  metabolism*
Transgenes
Chemical
Reg. No./Substance:
0/Fatty Acids, Unsaturated; EC 2.3.1.-/Acetyltransferases; EC 2.3.1.-/fatty acid elongases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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