| dc.description.abstracteng | Wax ester (WE) are neutral lipids, which consist of a fatty acid and a fatty alcohol moiety connected by an ester bond. WE are synthesized via two reactions from acyl-coenzyme A (CoA)/acyl carrier protein (ACP) substrates. In a first step, fatty acyl reductases (FAR) reduce acyl-CoA/ACP to form fatty alcohols. In the second step, fatty alcohols are esterified to another acyl-CoA/ACP by wax synthases (WS) to yield WE. Reflecting the diversity of acyl-chain substrates, a huge variety of WE exist in nature. Dependent on the chain length and further modifications such as desaturation of the acid and alcohol moieties, WE have diverse physicochemical properties, supporting their various functions in nature. WE may be deposited in the plant cuticle or on the human skin as protection agents and accumulate in bacteria and seeds of the desert shrub jojoba as carbon storage compounds. Due to their diverse properties, WE are used in various industrial applications. They are part of cosmetics, lubricants or candles. Until the banning of whale hunting, industrially used WE were obtained from spermaceti oil. Nowadays, WE are expensively extracted from jojoba seeds or are synthesized chemically from fossil fuels or plant oils. Attempts to cost-efficiently and environmentally-friendly synthesize tailor-made WE in suitable crop plants are made. However, further improvements regarding enzyme use, substrate availability and storage capacities of the sink tissue are necessary.
With the aim to improve tailor-made WE production in plants in terms of enzyme use and substrate availability, three main projects were conducted within this thesis. The first project dealt with the characterization of the fifth WS/acyl-CoA:diacylglycerol acyl transferase (WSD) from the bacterium Marinobacter aquaeolei (MaWSD5). Experiments revealed that the enzyme is a suitable candidate for WE production in plants. In vitro substrate specificity assays showed a broad substrate range of the enzyme and confirmed the lack of a side reaction towards TAG formation. Expression in Arabidopsis thaliana seeds together with a FAR from the same bacterium resulted in the production of WE consisting of long chain monoenoic moieties.
In the second project, a detailed structure-function analysis on the basis of the recently obtained crystal structure of WSD1 from the bacterium Acinetobacter baylyi (AbWSD1) was conducted. The identification of the diacylglycerol binding site and potential CoA binding residues provide now a basis for future protein engineering in order to generate WE producing enzymes with altered substrate specificities. A comparison of the AbWSD1 structure, co-crystallized with bound myristic acid, and the structure of WSD1 from Marinobacter aquaeolei revealed a structural rearrangement upon substrate binding and lead to the development of a substrate-binding model for WSD.
In the third project, the effect of cellular WE biosynthesis location was studied and a change in localization was established as a suitable tool to alter substrate availability in WE producing plants. Expression of different constructs consisting of FAR, WSD2 or WSD5 from Marinobacter aquaeolei with and without plastidial localization tag in A. thaliana seeds resulted in the formation of shorter and more saturated WE in plastids compared to cytosolic WE synthesis. | de |