Presentation Information
[P03-378]Engineering anaerobic metabolic branch points toward coproduction of wax ester and succinate in Euglena gracilis
○Rikuto Oishi1, Ryunosuke Katayama1, Mitsuhiro Ueda1, Tatsuji Sakamoto1, Masami Nakazawa1 (1. Osaka Metropolitan University (Japan))
Keywords:
microalgae,anaerobic metabolism,metabolic engineering,succinate accumulation,wax ester synthesis
[Purpose]
The microalgae Euglena gracilis produces wax esters (WEs) under anaerobic conditions by degrading the storage polysaccharide paramylon, while also accumulating succinate in the culture supernatant. Because part of the succinate is converted to succinyl-CoA and used for the synthesis of odd-chain WE components, this pathway may limit extracellular succinate accumulation. This study examined whether targeted modification of key metabolic branch points could increase succinate accumulation without substantially impairing overall WE synthesis.
[Method]
Wild-type E. gracilis strain Z and a succinyl-CoA synthetase α-subunit knockout strain (SCSα-KO) were used. Mitochondrial NADP+-dependent malic enzyme (ME) was suppressed by RNA interference (ME-RNAi), and the combined condition of SCSα-KO with ME-RNAi was also analyzed. Cells were cultivated aerobically under mixotrophic conditions and then subjected to 48 h of anaerobic incubation. Growth, paramylon content, WE accumulation and composition, and supernatant organic acids were analyzed. ME suppression was verified by semi-quantitative RT-PCR and enzyme activity assays.
[Results]
Aerobic growth, paramylon accumulation, and paramylon consumption during anaerobic incubation were generally comparable among the tested conditions. In the SCSα-KO strain, odd-chain WEs were almost completely lost, whereas total WE content remained at 56.7 μg per 106 cells, about 86% of the wild-type level (65.6 μg per 106 cells). Succinate accumulation increased from 12.1 to 31.6 μg per 106 cells. ME silencing increased succinate accumulation to 58.7 μg per 106 cells, while total WE content remained at 53.6 μg per 106 cells, or about 82% of the wild-type level. In the combined SCSα-KO ME-RNAi condition, total WE content was 50.5 μg per 106 cells, about 77% of the wild-type level, whereas succinate accumulation reached 107.4 μg per 106 cells, representing an 8.9-fold increase over the wild type. Malate and fumarate also increased under all modified conditions.
[Consideration]
These results suggest that carbon allocation at two anaerobic branch points strongly influences the balance between WE synthesis and organic acid secretion. SCSα disruption likely suppressed succinate utilization for odd-chain WE synthesis, whereas ME suppression likely promoted accumulation of reductive TCA-cycle intermediates by weakening the malate-to-pyruvate route. Their combination produced the highest succinate accumulation while retaining substantial WE synthesis.
[Conclusion]
Targeted modification of anaerobic metabolic branch points in Euglena gracilis increased succinate accumulation without severe loss of WE synthesis. In particular, the combination of SCSα deficiency and ME suppression markedly enhanced succinate production while maintaining substantial WE biosynthesis. These findings provide a basis for metabolic engineering aimed at the co-production of wax esters and succinate in E. gracilis.
The microalgae Euglena gracilis produces wax esters (WEs) under anaerobic conditions by degrading the storage polysaccharide paramylon, while also accumulating succinate in the culture supernatant. Because part of the succinate is converted to succinyl-CoA and used for the synthesis of odd-chain WE components, this pathway may limit extracellular succinate accumulation. This study examined whether targeted modification of key metabolic branch points could increase succinate accumulation without substantially impairing overall WE synthesis.
[Method]
Wild-type E. gracilis strain Z and a succinyl-CoA synthetase α-subunit knockout strain (SCSα-KO) were used. Mitochondrial NADP+-dependent malic enzyme (ME) was suppressed by RNA interference (ME-RNAi), and the combined condition of SCSα-KO with ME-RNAi was also analyzed. Cells were cultivated aerobically under mixotrophic conditions and then subjected to 48 h of anaerobic incubation. Growth, paramylon content, WE accumulation and composition, and supernatant organic acids were analyzed. ME suppression was verified by semi-quantitative RT-PCR and enzyme activity assays.
[Results]
Aerobic growth, paramylon accumulation, and paramylon consumption during anaerobic incubation were generally comparable among the tested conditions. In the SCSα-KO strain, odd-chain WEs were almost completely lost, whereas total WE content remained at 56.7 μg per 106 cells, about 86% of the wild-type level (65.6 μg per 106 cells). Succinate accumulation increased from 12.1 to 31.6 μg per 106 cells. ME silencing increased succinate accumulation to 58.7 μg per 106 cells, while total WE content remained at 53.6 μg per 106 cells, or about 82% of the wild-type level. In the combined SCSα-KO ME-RNAi condition, total WE content was 50.5 μg per 106 cells, about 77% of the wild-type level, whereas succinate accumulation reached 107.4 μg per 106 cells, representing an 8.9-fold increase over the wild type. Malate and fumarate also increased under all modified conditions.
[Consideration]
These results suggest that carbon allocation at two anaerobic branch points strongly influences the balance between WE synthesis and organic acid secretion. SCSα disruption likely suppressed succinate utilization for odd-chain WE synthesis, whereas ME suppression likely promoted accumulation of reductive TCA-cycle intermediates by weakening the malate-to-pyruvate route. Their combination produced the highest succinate accumulation while retaining substantial WE synthesis.
[Conclusion]
Targeted modification of anaerobic metabolic branch points in Euglena gracilis increased succinate accumulation without severe loss of WE synthesis. In particular, the combination of SCSα deficiency and ME suppression markedly enhanced succinate production while maintaining substantial WE biosynthesis. These findings provide a basis for metabolic engineering aimed at the co-production of wax esters and succinate in E. gracilis.
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