Metabolic engineering of medium-chain fatty acid biosynthesis in Nicotiana benthamiana plant leaf lipids

Abstract

Various research groups are investigating the production of oil in non-seed biomass such as leaves. Recently, high levels of oil accumulation have been achieved in plant biomass using a combination of biotechnological approaches which also resulted in significant changes to the fatty acid composition of the leaf oil. In this study, we were interested to determine whether medium-chain fatty acids (MCFA) could be accumulated in leaf oil. MCFA are an ideal feedstock for biodiesel and a range of oleochemical products including lubricants, coatings, and detergents. In this study, we explore the synthesis, accumulation, and glycerolipid head-group distribution of MCFA in leaves of Nicotiana benthamiana after transient transgenic expression of C12:0-, C14:0-, and C16:0-ACP thioesterase genes. We demonstrate that the production of these MCFA in leaf is increased by the co-expression of the WRINKLED1 (WRI1) transcription factor, with the lysophosphatidic acid acyltransferase (LPAAT) from Cocos nucifera being required for the assembly of tri-MCFA TAG species. We also demonstrate that the newly-produced MCFA are incorporated into the triacylglycerol of leaves in which WRI1 + diacylglycerol acyltransferase1 (DGAT1) genes are co-expressed for increased oil accumulation.

Properties and Biotechnological Applications of Acyl‐CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae

“…The combined overexpression of cDNA encoding DGAT1, WRI, and oleosin, respectively, is a very effective approach for increasing leaf TAG, driving oil accumulation to more than 15% dry weight in N. tabacum (Vanhercke et al, ). Increased incorporation of medium‐chain fatty acids into TAG has also been obtained in tobacco leaves through overexpression of DGAT1 together with other genes that can increase the flux of medium‐chain fatty acids in the pathway (Reynolds et al, , ). Increased oil content in vegetative tissues was also obtained in Arabidopsis (Kelly et al, ; Winichayakul et al, ), Saccharum spp.…”

WRINKLED1 Rescues Feedback Inhibition of Fatty Acid Synthesis in Hydroxylase-Expressing Seeds

“…A loss-of-function mutation, wri1, in Arabidopsis causes an 80% reduction in the level of FA in seeds, which leads to a wrinkled appearance (Focks and Benning, 1998;Cernac and Benning, 2004). Both in seeds and in vegetative tissues, overexpression of WRI1 leads to an increase in the level of FA accumulating in TAG (Cernac and Benning, 2004;Shen et al, 2010;Sanjaya et al, 2011;Kelly et al, 2013;Vanhercke et al, 2013Vanhercke et al, , 2014van Erp et al, 2014;Wu et al, 2014;Grimberg et al, 2015;Reynolds et al, 2015;Zale et al, 2016). WRI1 overexpression elevates the transcript level of its target genes involved in FA synthesis (Maeo et al, 2009;To et al, 2012), and this is assumed to lead to an increase in protein level and enzyme activity.…”

NMT1 and NMT3 N-Methyltransferase Activity Is Critical to Lipid Homeostasis, Morphogenesis, and Reproduction

“…Chloroform extracts of lipids from plant tissue were dried under nitrogen and resuspended in 989 chloroform to 100 mg/ml, then diluted to 1 mg/ml in butanol:methanol (1:1, v/v) and 990 analyzed on by liquid chromatography-mass spectrometry (LC-MS), based on previously 991 described methods (Reynolds et al, 2015). Briefly, a Waters BEH C8 (100 mm x 2.1 mm, 2.7 exported for further analysis using the dplyr and ggplot2 packages from R using RStudio 1020 (Horton and Klenman, 2015).…”