RuBisCO-based CO2 fixation improves glutamate production in Corynebacterium glutamicum

Aiying Wei^1,2Jingui Liu¹Yulin Tang¹Gang Meng²Chunguang Zhao²Houbo Su²Heyun Wu^1,3Qian Ma^1,3Xixian Xie^1,3*

• ¹College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China
• ²Ning Xia Eppen Biotech Co., Ltd.,, Ningxia, 750100, China
• ³Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, Tianjin, 300457, China

Background and introduction: Efficiently harnessing CO2 for the bioproduction of chemicals stands as an important way to mitigate CO2 emissions and actively advance the achievement of carbon neutrality. Drawing inspiration from the natural Calvin-Benson-Bassham (CBB) cycle for CO2 fixation, the heterologous introduction of phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) into microbial cell factories emerges as a highly promising method for fully harnessing CO2 for bioproduction purposes. Methods: In this study, we engineered the industrial glutamate-hyperproducing strain Corynebacterium glutamicum YPGlu001 by introducing a heterologous RuBisCO-PRK pathway. Two metabolic configurations were evaluated: a "replacement" strategy, which blocked native glycolytic and pentose phosphate pathway (PPP) fluxes (via Δgap, ΔgapX, Δpgk, and Δzwf) to force carbon through the CBB shunt; and a "complementation" strategy, where the CO2-fixation pathway supplemented the native central metabolism. Pathway performance was optimized through promoter engineering (Ptac, PH30, Pfba, PgroES) and adaptive laboratory evolution (ALE) under 10% CO2 enrichment. Results: Comparative analysis revealed that the "replacement" strategy severely impaired cell growth and glutamate synthesis, with ALE failing to restore the desired production levels. In contrast, the "complementation" strategy significantly enhanced metabolic performance. The optimized strain GluE014 exhibited superior carbon-to-product conversion, achieving a glutamate titer of 196.78 g/L in a 5 L fed-batch fermenter within 30 hours. This represents a 13.94% increase in titer and an 11.55% improvement in glucose-based yield compared to the parental strain. Furthermore, the engineered strain demonstrated improved carbon economy, reducing glucose consumption by 5.24% while maintaining high productivity. Conclusions: This work demonstrates that "replenishing" native metabolism with a CO2-fixation shunt is more effective than "replacing" essential pathways in industrial C. glutamicum. By successfully integrating heterologous CO2 assimilation with robust industrial fermentation, this study provides a scalable and efficient blueprint for developing next-generation, carbon-negative microbial cell factories.