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

Intensification of media components can effectively improve biochemical production by reinforcing the metabolic capacity of engineered strains. Here, we present a citrate-enabled medium intensification strategy for sustainable l-theanine biomanufacturing in Corynebacterium glutamicum. Recombinant strains expressing 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.