Engineering Anaerobic Metabolic Branch Points to Control Carbon Allocation Between Wax Ester Synthesis and Succinate...

Under anaerobic conditions, Euglena gracilis degrades paramylon and synthesizes wax esters (WEs) while accumulating succinate in the culture supernatant. Succinate can either be converted to succinyl-CoA by succinyl-CoA synthetase (SCS) and enter the propionyl-CoA biosynthetic pathway or accumulate extracellularly. Propionyl-CoA serves as a primer for odd-chain WE synthesis. Prior research revealed that knockout (KO) of the SCS α-subunit (SCSα) nearly eliminated odd-chain WE synthesis while preserving overall WE accumulation. In the present study, the effects of targeted modifications at anaerobic metabolic branch points on carbon allocation between WE synthesis and succinate accumulation were examined. SCSα KO increased succinate accumulation and markedly reduced odd-chain WEs while largely maintaining total WE levels. Silencing mitochondrial NADP⁺-dependent malic enzyme (ME), which affects carbon allocation at the malate branch point between WE synthesis and the fumarate-to-succinate pathway, also increased succinate accumulation, although total WE accumulation decreased only slightly. Combining ME silencing with SCSα KO increased succinate accumulation to 8.9-fold that of the wild type while retaining more than 75% of the WT total WE level. These findings demonstrate that targeted modification of anaerobic metabolic branch points can redirect carbon allocation toward succinate accumulation without abolishing WE biosynthesis in E. gracilis.