Abstract:
L-pipecolic acid (L-PA) is a non-proteinogenic amino acid, however, it is an important precursor and a key ingredient of many pharmaceutically compound syntheses such as immunosuppressants and anesthetics. The production of amino acids via fermentation of microorganisms are getting attention, one of the widely used is an engineered E. coli. L-PA production in the engineered E. coli uses L-lysine in its cell as a substrate. This research aimed to increase L-PA production by thrA knockout. The thrA encodes homoserine dehydrogenase I, the enzyme which draws the intermediate in L-lysine biosynthesis pathway to synthesize L-threonine. The group II intron insertion was used for disruption of thrA by specific insertion at the target gene. The capability of group II intron insertion for thrA in E. coli (E. coli BL21(DE3) ΔthrA) was 65%. Moreover, pE22-LPC*D* was constructed and transformed into E. coli BL21(DE3) (namely W-LPCD) and E. coli BL21(DE3) ΔthrA (namely KO-LPCD). pE22-LPC*D* consisted of lysdh (encoding for lysine 6-dehydrogenase) from Acromobacter denitrificans, proC (encoding for pyrroline-5-carboxylate reductase) from Bacillus cereus ATCC 11778, and homologous lysC* and dapA* which encode for lysine feedback resistant aspartokinase and dihydrodipicolinate synthase, respectively. The highest L-PA production by KO-LPCD was 0.57 g/L when it was cultured in Ying medium containing glycerol as a carbon source at 198 hours after induction with 0.1 mM IPTG. The specific L-PA production was 0.049 g/g WCW, which was 1.8-fold of that obtained from W-LPCD.
