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-pycA cgl ) mianly aggregated in the state of inclusion body. Then a low copy plasmid pWSK29 was employed for pycA bsu 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.
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Wang, J. et al. (2018). Effects of Heterologous Pyruvate Carboxylase Expression on Synthesis of l-Threonine in Escherichia coli . In: Liu, H., Song, C., Ram, A. (eds) Advances in Applied Biotechnology. ICAB 2016. Lecture Notes in Electrical Engineering, vol 444. Springer, Singapore. https://doi.org/10.1007/978-981-10-4801-2_14
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DOI: https://doi.org/10.1007/978-981-10-4801-2_14
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