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Title: Reducing isozyme competition increases target fatty acid accumulation in seed triacylglycerols of transgenic Arabidopsis

item VAN ERP, HARRIE - Washington State University
item Shockey, Jay
item ZHANG, MENG - Michigan State University
item ADHIKARI, NEIL - Michigan State University
item BROWSE, JOHN - Michigan State University

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/3/2015
Publication Date: 3/4/2015
Citation: Van Erp, H., Shockey, J., Zhang, M., Adhikari, N.D., Browse, J. 2015. Reducing isozyme competition increases target fatty acid accumulation in seed triacylglycerols of transgenic Arabidopsis. Plant Physiology. 168:36-46.

Interpretive Summary: Manipulation of transgenic oilseed fatty acid content has progressed only very slowly in recent years. Many reasons might explain why this is so. One reason is that the scientific community has not yet fully developed the necessary sets of genes to stack into engineered crops. However, one other possibility is that similar enzymes present in the host plant species might outcompete the enzymes introduced through the transformation process. In this study we addressed this question by simultaneously introducing critical genes from either castor bean or tung tree, while also completely or partially silencing the enzyme activities in the host species. Higher levels of novel fatty acid products were produced when this combined approach was used. This dual approach may therefore be of great use to the biotechnology community going forward.

Technical Abstract: One goal of green chemistry is the production of industrially useful fatty acids (FAs) in crop plants. We focus on the engineering of industrial FAs, specifically hydroxy fatty acids (HFA) and conjugated polyenoic fatty acids (a-eleostearic acid, ESA), using Arabidopsis (Arabidopsis thaliana) as a model. These FAs are found naturally in seed oils of castor (Ricinus communis) and tung (Vernicia fordii), respectively, and are used for the production of lubricants, nylon and paints. Transgenic oils typically contain less target FA than produced in the exotic species. We hypothesize that isozyme competition for substrates limits the accumulation of novel FAs in Arabidopsis seeds. This hypothesis was tested by introducing a mutation in AtDGAT1 in a line expressing castor FA hydroxylase (RcFAH12) and diacylglycerol:acyltransferase2 (RcDGAT2) in its seeds. This led to a 4% increase in HFA levels. Expression of castor phospholipid:diacylglycerol acyltransferase1A (PDAT1A) in this line increased the level of HFA by an additional 4%. To determine if our observations are more generally applicable, we investigated if isozyme competition influenced the production of ESA. Expression of tung FA conjugase (FADX, (Dyer et al., 2002)) produced ~7.5% ESA in seed lipids. Coexpression of VfDGAT2 increased ESA levels to ~11%. Overexpression of VfDGAT2, combined with suppression of AtDGAT1 increased ESA accumulation to 14-15%. This research indicates that isozyme competition is a limiting factor in the engineering of unusual FAs in heterologous plant systems, and that reduction of competition through mutation and RNA suppression, may be a useful component of seed metabolic engineering strategies.