Alteration of Wax Ester Content and Composition in Euglena gracilis with Gene Silencing of 3‐ketoacyl‐CoA Thiolase Isozymes

Abstract

Euglena gracilis produces wax ester under hypoxic and anaerobic culture conditions with a net synthesis of ATP. In wax ester fermentation, fatty acids are synthesized by reversing beta-oxidation in mitochondria. A major species of wax ester produced by E. gracilis is myristyl myristate (14:0-14:0Alc). Because of its shorter carbon chain length with saturated compounds, biodiesel produced from E. gracilis wax ester may have good cold flow properties with high oxidative stability. We reasoned that a slight metabolic modification would enable E. gracilis to produce a biofuel of ideal composition. In order to produce wax ester with shorter acyl chain length, we focused on isozymes of the enzyme 3-ketoacyl-CoA thiolase (KAT), a condensing enzyme of the mitochondrial fatty acid synthesis pathway in E. gracilis. We performed a gene silencing study of KAT isozymes in E. gracilis. Six KAT isozymes were identified in the E. gracilis EST database, and silencing any three of them (EgKAT1-3) altered the wax ester amount and composition. In particular, silencing EgKAT1 induced a significant compositional shift to shorter carbon chain lengths in wax ester. A model fuel mixture inferred from the composition of wax ester in EgKAT1-silenced cells showed a significant decrease in melting point compared to that of the control cells.

The Mitochondrion of Euglena gracilis

“…C28 wax esters were dominant in the control cells and in thiolase-silenced cells C26 (isoform 1) and C27 (isoform 2). Gene silencing of these two isoforms significantly increased the amount and the ratio of wax esters with an acyl chain length shorter than C26 (Nakazawa et al 2015). The study suggests different roles for each isoenzyme in hypoxic wax ester production (Nakazawa et al 2015).…”

“…A recent study showed that gene silencing of this enzyme altered both the wax ester content and the wax ester composition in E. gracilis, inducing a significant compositional shift to shorter carbon chain lengths in wax esters (Nakazawa et al 2015). Six putative 3-ketoacyl-CoA thiolase isoenzymes were found in an E. gracilis EST database (O'Brien et al 2007), but only the gene silencing of three of them had an effect on wax esters.…”

“…Gene silencing of these two isoforms significantly increased the amount and the ratio of wax esters with an acyl chain length shorter than C26 (Nakazawa et al 2015). The study suggests different roles for each isoenzyme in hypoxic wax ester production (Nakazawa et al 2015). Isoenzyme 3 might be a short-and medium-chain-specific enzyme important for the hypoxic synthesis of wax esters from paramylon, while isoforms 1 and 2 might be medium-and long-chain-specific in the acyl-CoA production in mitochondrial fatty acid synthesis (Nakazawa et al 2015).…”

Wax Ester Fermentation and Its Application for Biofuel Production

“…This isozyme was highly active with both crotonyl-CoA and trans-2-hexenoyl-CoA (C 6 ), and both NADH and NADPH were suitable electron donors. In addition to TER, 3-ketoacylCoA thiolase isozymes that catalyze the condensation reaction of acyl-CoA and acetyl-CoA in the mitochondrial fatty acid synthesis, have been studied in Euglena (Nakazawa et al 2015) (for details, see Sect. 13.4.3).…”

“…A likely strategic point for increased production of wax esters in Euglena is thus elevating the TER activity by genetic engineering. Nakazawa et al (2015) reported that at least three isozymes of 3-ketoacyl-CoA thiolase (EgKAT1-3) participated in mitochondrial fatty acid synthesis in the wax ester fermentation. It was also shown that silencing of EgKAT3 led to a decreased wax ester content while silencing of EgKAT1 and EgKAT2 led to a significant shift to shorter carbon chain lengths in the wax esters.…”