Abstract
The beta-ketoacyl-ACP synthase II (KASII) is an enzyme in fatty acid biosynthesis, catalyzing the elongation of 16:0-acyl carrier protein (ACP) to 18:0-ACP in plastids. Mutations in KASII genes in higher plants can lead to lethality, which makes it difficult to utilize the gene for lipid metabolic engineering. We demonstrated previously that transient expression of plastid-directed fatty acyl reductases and wax ester synthases could result in different compositions of wax esters. We hypothesized that changing the ratio between C16 (palmitoyl-compounds) and C18 (stearoyl-compounds) in the plastidic acyl-ACP pool by inhibition of KASII expression would change the yield and composition of wax esters via substrate preference of the introduced enzymes. Here, we report that transient inhibition of KASII expression by three different RNAi constructs in leaves of N. benthamiana results in almost complete inhibition of KASII expression. The transient RNAi approach led to a shift of carbon flux from a pool of C18 fatty acids to C16, which significantly increased wax ester production in AtFAR6-containing combinations. The results demonstrate that transient inhibition of KASII in vegetative tissues of higher plants enables metabolic studies towards industrial production of lipids such as wax esters with specific quality and composition.
Highlights
Oil crops are of great interest since they can provide a sustainable production of high-value oleochemicals such as wax esters and fatty alcohols for the chemical industry with similar hydrocarbon structures to those of conventional petrochemical products
In order to test whether simultaneous inhibition of the two ketoacyl-acyl carrier protein (ACP) synthase II (KASII) genes was possible, three different RNAi constructs were made towards different regions of KASII using intron-spliced hairpin technology[28]
The results presented here provide evidence that hairpin-RNAi targeting the KASII genes in Nicotiana benthamiana leaf tissues makes more palmitic acid available for metabolism of complex products and, as a consequence, elevates the C16/C18 ratio in wax esters
Summary
Oil crops are of great interest since they can provide a sustainable production of high-value oleochemicals such as wax esters and fatty alcohols for the chemical industry with similar hydrocarbon structures to those of conventional petrochemical products. The fatty acid 16:0-ACP is a substrate for KASII and FATB, and for SAD17 and lysophosphatidyl acyltransferase (LPAAT)[18] These enzymes together are important for determination of the fatty acid pool available to the lipid biosynthetic machinery in the cytosol. The second stage of the enzymatic process is conversion of an activated fatty acid to a fatty alcohol by the function of a fatty acyl reductase (FAR) enzyme. Our aim was to demonstrate that modulation of KASII expression is possible in plant vegetative tissues This provides a rational experimental approach for increasing levels of C16:0 and decreasing levels of C18:0 in the metabolic engineering of wax ester synthesis for improvement of the specific composition and quality of wax esters for industrial purposes
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