Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis.
Bacillus subtilis (B. subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins. In this study, a shuttle vector was constructed by fusion of Staphylococcus aureus (S. aureus) plasmid pUB110 with Escherichia coli (E. coli) plasmid pUC18 and used for the expression of nattokinase in B. subtilis. The pUB110/pUC-based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions. An initial attempt was made to eliminate the homologous site in the plasmid, whereas the stability of the resulting plasmid was not improved. In an alternative way, the pUC18-derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E. coli. As a consequence, the pUB110/R6K-based plasmid displayed full structural stability, leading to a high-level production of recombinant nattokinase in the culture broth. This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid. Moreover, 2-fold higher nattokinase production was obtained by B. subtilis strain carrying the pUB110/R6K-based plasmid as compared to the cell with the pAMbeta1-derived vector, a plasmid known to have high structural stability. Overall, it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B. subtilis.
Chen
Po Ting
PT
Department of Chemical Engineering, Feng Chia University, 100 Wenhwa Road, Taichung 40724, Taiwan.
Chiang
Chung-Jen
CJ
Chao
Yun-Peng
YP
D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013381:Subtilisins / Q000737:chemistry*
D017931:DNA Primers; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D004247:DNA; D013381:Subtilisins; C053771:nattokinase