Effects of heterologous pyruvate carboxylase expression on synthesis of L-threonine in Escherichia coli

Abstract

The carboxylation reaction catalyzed by pyruvate carboxylase (Pyc) is the major approach to supply oxaloacetate in many bacteria, but this enzyme is absent in Escherichia coli. The SDS-PAGE results displayed the active expression of Pyc from Bacillus subtilis (pTrc99a-pycA) in E. coli THRD, a l-threonine peoducer, whereas that from Corynebacterium glutamicum (pTrc99a-pycAcgl) mianly aggregated in the state of inclusion body. Then a low copy plasmid pWSK29 was employed for pycAbsu expression in THRD to investigate the influence of expression level on l-threonine fermentation, and the RT-qPCR results revealed that the relative expression level in pTrc99a-pycA was 3.8 times of that in pWSK29-pycA. The shake-flask culture of THRD/pWSK29-pycA resulted in a titer of 46.09 g/L l-threonine and a yield of 30.72%, which was 12.28 and 12.24% higher than that of the control (THRD/pWSK29), respectively. The titer of l-threonine in THRD/pTrc99a-pycA culture reached 35.91 g/L, 10.36% lower than that obtained from the control (THRD/pTrc99a); however, the biomass increased by 31.69%. The results showed that appropriate expression of Pyc was conducive to the synthesis of l-threonine. Therefore, the strain THRD pykF::pycA was constructed with chromosomal integration of pycA in the locus of pykF, but the fermentation results of this strain did not show significant difference compared with THRDΔpykF; and the possible explanations were discussed.