Enhancing the production of L-proline in recombinant Escherichia coli BL21 by metabolic engineering.

L-proline is widely used in the fields of food, medicine and agriculture, and is also an important raw material for the synthesis of trans-4-hydroxy-L-proline. In this study, enhancing the production of L-proline by metabolic engineering was investigated. Three genes, proB, proA and proC, were introduced into Escherichia coli BL21 by molecular biology technology to increase the metabolic flow of L-proline from glucose. The genes putP and proP related to the proline transfer were knocked out by CRISPR/Cas9 gene editing technology to weaken the feedback inhibition of proB to increase the production of L-proline. The fermentation curves of the engineered strain at different glucose concentrations were determined, and a glucose concentration of 10 g/L was chosen to expand the batch culture to 1 L shake flask. Ultimately, through these efforts, the titer of L-proline reached 832.19 mg/L in intermittent glucose addition fermentation in a 1 L shake flask.