Magdalena Miklaszewska

Functional characterisation of algal wax ester biosynthetic enzymes and evaluation of their potential for wax ester production in microalgae

With an increasing world population, demand on industrial feedstocks and predicted effects of global warming, there is a strong need for renewable materials. Algae convert sunlight into chemical energy and biomaterials by fixing CO2 and therefore may provide a clean solution for a sustainable bioeconomy.

Wax esters, long-chain esters of fatty acids and fatty alcohols, are produced by a wide variety of organisms. They fulfil numerous biological functions, including energy storage, buoyancy regulation and protection against water loss, UV light and pathogens. In industry, wax esters are used as components of high-pressure lubricants, pharmaceuticals, cosmetics and printing inks. Large-scale production of wax esters, based on chemical or enzymatic processes, is expensive and has a negative environmental impact. Comparably small amounts of natural wax esters can be obtained from Simmondsia chinensis (Jojoba). Therefore, there is a need to establish an attractive alternative platform for biotechnological production of wax esters from renewable resources.

Fatty acyl reductase (FAR) and wax synthase (WS) are two main types of enzymes involved in the wax ester biosynthesis. FAR produces fatty alcohols from fatty acyls, then WS catalyses the esterification reaction of a fatty acyl with a fatty alcohol. So far, a large number of FARs and WSs from different organisms have been identified and characterised. However, there are no reports on isolation of the wax ester-synthesising enzymes of algal origin. My preliminary studies showed wax ester accumulation in Chlamydomonas reinhardtii cells cultured under nitrogen deprivation. In addition, the analysis of the algal genome sequences revealed the presence of putative enzymes involved in the wax esters biosynthesis in the members of the Chlorophyta phylum, including C. reinhardtii.

The aim of this project is to characterize enzymes involved in wax ester biosynthesis in the model green alga C. reinhardtii and reveal the physiological role of wax esters in algae. Next, I will use this knowledge to boost wax ester production in green algae using state-of-the-art genetic engineering and synthetic biological tools. This is a first attempt to characterise putative FARs and WSs of algal origin and explore their suitability for wax ester production in genetically modified algae. The project will also elucidate the yet unknown physiological function of wax esters in C. reinhardtii.

The research plan consists of three work packages. WP1 includes cloning and heterologous expression of genes encoding C. reinhardtii FARs and WSs and determination of activity and specificity of these enzymes in in vivo and in vitro conditions using yeast and transient expression in Nicotiana benthamiana leaves. WP2 will focus on analysing content and composition of wax esters synthesised in C. reinhardtii cells under different conditions and studies on C. reinhardtii strains with disrupted genes encoding putative FARs and WSs. In WP3 selected strains of C. reinhardtii will be genetically engineered towards increased wax ester synthesis using already available FARs and WSs of different origin as well as newly characterised enzymes from C. reinhardtii.

This project will be an important contribution to the knowledge about algal enzymes involved in the wax ester synthesis. The results of the project will provide a valuable insight into lipid metabolism in algae and will facilitate a better understanding of wax ester production in this promising group of organisms. The identification of algal FARs and WSs and the establishment of the reliable approach for their comprehensive biochemical characterisation may lead to the discovery of enzymes involved in the wax ester synthesis in other members of the Chlorophyta phylum. The results of the project could be also important for the development of cost-effective and environmental friendly methods of large-scale fatty alcohol and wax ester production in algae.