Expanding metabolic pathway for de novo biosynthesis of the chiral pharmaceutical intermediate L-pipecolic acid in Escherichia coli.

BACKGROUND

The six-carbon circular non-proteinogenic compound L-pipecolic acid is an important chiral drug intermediate with many applications in the pharmaceutical industry. In the present study, we developed a metabolically engineered strain of Escherichia coli for the overproduction of L-pipecolic acid from glucose.

RESULTS

The metabolic pathway from L-lysine to L-pipecolic acid was constructed initially by introducing lysine cyclodeaminase (LCD). Next, L-lysine metabolic flux from glucose was amplified by the plasmid-based overexpression of dapA, lysC, and lysA under the control of the strong trc promoter to increase the biosynthetic pool of the precursor L-lysine. Additionally, since the catalytic efficiency of the key enzyme LCD is limited by the cofactor NAD, the intracellular pyridine nucleotide concentration was rebalanced by expressing the pntAB gene encoding the transhydrogenase, which elevated the proportion of LCD with bound NAD and enhanced L-pipecolic acid production significantly. Further, optimization of Fe and surfactant in the fermentation process resulted in 5.33 g/L L-pipecolic acid, with a yield of 0.13 g/g of glucose via fed-batch cultivation.

CONCLUSIONS

We expanded the metabolic pathway for the synthesis of the chiral pharmaceutical intermediate L-pipecolic acid in E. coli. Using the engineered E. coli, a fast and efficient fermentative production of L-pipecolic acid was achieved. This strategy could be applied to the biosynthesis of other commercially and industrially important chiral compounds containing piperidine rings.