[3S04e] 11. The Society for Biotechnology - Part 3

6:15 PM - 6:30 PM JST(9:15 AM - 9:30 AM UTC)

[3Plant-01-KL] Genome editing of the β-oxidation reversal pathway achieves stable modification of anaerobic wax ester composition in Euglena gracilis

Sakura Nagamine¹, Rikuto Oishi¹, ○Masami Nakazawa¹ (1. Osaka Metropolitan University (Japan))

Keywords:

Genome-editing,microalgae,Euglena gracilis,wax ester

Euglena gracilis accumulates large amounts of wax esters under anaerobic conditions (previously termed “wax ester fermentation”). This anaerobic wax ester synthesis depends on anaerobic respiration coupled to mitochondrial fatty acid synthesis via the β-oxidation reversal pathway. In this pathway, acetyl-CoA or propionyl-CoA is used as a primer, and sequential reactions of the four β-oxidation enzymes elongate acyl chains using acetyl-CoA as the C2 donor. The resulting wax esters are mainly composed of saturated fatty acids and fatty alcohols with acyl-chain lengths of approximately C11:0–C16:0, with C14:0–C14:0 alcohol as a major component, making them attractive as potential feedstocks for biofuels such as biodiesel. In addition, the presence of odd-chain fatty acids and alcohols suggests opportunities for unique applications beyond conventional bio-based resources. Accordingly, tailoring wax ester composition can both optimize lipid properties for target applications and broaden the utility of E. gracilis wax esters as chemical feedstocks.

Previously, transient shifts in wax ester composition were achieved using RNA interference during pathway elucidation. However, controlling composition for target applications requires stable, repeatable genetic changes. Here, we pursued genome editing-based engineering to enable stable modification and to identify actionable control points for composition tuning. Specifically, we focused on isozymes of acyl-CoA dehydrogenase (ACD) and 3-ketoacyl-CoA thiolase (KAT), which are expected to influence acyl-CoA intermediates and chain-length outcomes during anaerobic elongation.

Genome editing-based knockout of selected ACD and KAT isozyme genes produced clear, isozyme-dependent shifts in anaerobic wax ester chain-length distributions. Knockout of a short-chain-type ACD isozyme promoted accumulation of longer-chain wax esters, whereas disruption of long-chain-type ACD and KAT isozymes shifted products toward shorter-chain components. These stable phenotypes demonstrate that targeted genome editing of β-oxidation reversal isozymes provides a practical route for rationally tuning anaerobic wax ester composition in E. gracilis, supporting tailored bioproduction and expanded chemical applications.