Efficient Escherichia coli platform for L-proline biosynthesis via systematic metabolic engineering

Despite its prominent industrial use for L-proline production, microbial fermentation by Corynebacterium glutamicum achieves a suboptimal yield of glucose conversion, far below the theoretical maximum of 0.63 g/g. This study aimed to develop an efficient engineered Escherichia coli chassis to produce L-proline from glucose by addressing the rate-limiting enzymatic step and high glucose conversion. The γ-glutamyl kinase encoding gene ( proB ) was screened by directed evolution, and the catalytic efficiency of the Sc ProB** variant toward L-glutamate increased 2.75-fold. After channeling 2-oxoglutarate flux and supplying sufficient NADPH, L-proline production reached 86.27 ± 2.58 g/L, with a 0.33 ± 0.01 g/g glucose yield in 10-L fed-batch fermentation. Further increasing the flux through central carbon metabolism by implementing a non-PTS glucose uptake system, amplifying the anaplerotic reaction, and redirecting the flux away from the glyoxylate cycle, the resulting strain BP23 achieved 119.89 ± 5.66 g/L L-proline, with a productivity of 1.93 ± 0.03 g/L/h. The glucose conversion yield improved to 0.49 ± 0.01 g/g after 62 h of fed-batch fermentation, indicating that the E. coli strain is a promising workhorse for producing L-proline with high glucose conversion.