Citrate-enabled process intensification reinforces energy and redox metabolism for sustainable bioproduction using Corynebacterium glutamicum

Abstract

heterologous theanine synthases were constructed using a strain expressing L-glutamate-L-cysteine ligase from Escherichia coli (THE-6) to achieve high theanine production. To establish a sustainable process, renewable biosugars derived from hydrothermally pretreated sweet sorghum bagasse were used as fermentation feedstock. Cultivation with biosugar-based media significantly enhanced theanine titers compared to cultivation with analytical-grade sugars. Integrated metabolic profiling using biosugars as natural test cocktails identified citrate as a key medium-derived factor that enhanced productivity. Controlled citrate supplementation improves theanine production. Integrated metabolomic-transcriptional analyses suggest that citrate accelerates TCA cycledriven oxidative metabolism and elevates intracellular ATP availability, thereby enhancing precursor utilization toward theanine biosynthesis. Under carbon-equivalent conditions, citrate supplementation increased theanine titers by up to approximately 1.5-fold and improved carbon yields by approximately 10-30% compared to glucose-only controls. Notably, the citrate-enabled intensification effect extended beyond ATP-intensive theanine biosynthesis to NADH-dependent pathways, including lysine, glutaric acid, and 5-hydroxyvaleric acid production, resulting in approximately 1.2- to 2.5-fold increases in product titers depending on the pathway configuration, consistent with strengthened cellular redox metabolism. Overall, the proposed biosugar-guided, integrated metabolomics-transcriptional analysis approach revealed effective media intensification factors and demonstrated that citrate supplementation can broadly strengthen cellular energy and redox metabolism. These findings support citrate-enabled medium intensification as a complementary energy- and redox-reinforcement heterologous theanine synthases were constructed using a strain expressing L-glutamate-L-cysteine ligase from Escherichia coli (THE-6) to achieve high theanine production. To establish a sustainable process, renewable biosugars derived from hydrothermally pretreated sweet sorghum bagasse were used as fermentation feedstock. Cultivation with biosugar-based media significantly enhanced theanine titers compared to cultivation with analytical-grade sugars. Integrated metabolic profiling using biosugars as natural test cocktails identified citrate as a key medium-derived factor that enhanced productivity. Controlled citrate supplementation improves theanine production. Integrated metabolomic-transcriptional analyses suggest that citrate accelerates TCA cycle-driven oxidative metabolism and elevates intracellular ATP availability, thereby enhancing precursor utilization toward theanine biosynthesis. Under carbon-equivalent conditions, citrate supplementation increased theanine titers by up to approximately 1.5-fold and improved carbon yields by approximately 10-30% compared to glucose-only controls. Notably, the citrate-enabled intensification effect extended beyond ATP-intensive theanine biosynthesis to NADH-dependent pathways, including lysine, glutaric acid, and 5-hydroxyvaleric acid production, resulting in approximately 1.2-to 2.5-fold increases in product titers depending on the pathway configuration, consistent with strengthened cellular redox metabolism. Overall, the proposed biosugar-guided, integrated metabolomics-transcriptional analysis approach revealed effective media intensification factors and demonstrated that citrate supplementation can broadly strengthen cellular energy and redox metabolism. These findings support citrate-enabled medium intensification as a complementary energy-and redox-reinforcement strategy alongside conventional metabolic engineering.