Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum.

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Growing global demand for biobased polymers necessitates improved microbial production of precursors like glutarate.
• Current production hosts require optimization for higher titers, yields, and reduced bioprocess duration.

Purpose of the Study:

• To enhance glutarate production in *C. glutamicum* using adaptive laboratory evolution.
• To investigate the link between faster growth and improved glutarate production via flux enforcement.

Main Methods:

• Engineered *C. glutamicum* with flux enforcement by deleting the *gdh* gene to couple growth to glutarate production.
• Applied adaptive laboratory evolution through serial dilution to select for faster-growing mutants.
• Utilized genome sequencing and reverse genetics to identify causal mutations.
• Performed bioreactor-scale fermentation (batch and fed-batch) and optimized downstream reactive extraction.

Main Results:

• Achieved a twofold increase in volumetric productivity (0.18 g L^-1 h^-1) in evolved mutants compared to the parental strain.
• Identified an amino acid exchange in L-glutamic acid-2-oxoglutarate aminotransferase as causal for accelerated glutarate production.
• Demonstrated stable performance at 2 L scale, reaching a titer of 22.7 g L^-1, yield of 0.23 g g^-1, and productivity of 0.35 g L^-1 h^-1.
• Optimized reactive extraction for glutarate recovery with high yields (58% and 99%).

Conclusions:

• Flux enforcement enables selection of faster-growing and producing strains via adaptive laboratory evolution.
• Genome sequencing and reverse genetics are effective for identifying causal mutations in metabolic engineering.
• This approach provides a rational strategy for improving microbial production of chemicals like glutarate.