Background: A Bacillus subtilis (B. subtilis) promoter, the vegI promoter (vegIP), has been well studied and shown to be a strong promoter for directing gene expression in gram-positive bacteria. However, when vegIP is harbored in a shuttle vector with a high plasmid copy number (PCN), pM2vegI, it is untransformable in B. subtilis. The plasmid has been found to be transformable in B. subtilis only when a spontaneous point or deletion mutation is introduced at the -11 position of vegIP. My thesis study aims to solve the intransformability issue associated with the use of wild-type vegIP in B. subtilis for the production of recombinant proteins, such as the endoglucanase of Cellulomonas fimi (C. fimi), CenA.

Methodology/Principal Findings: I hypothesize that the intransformability...[ Read more ]

Background: A Bacillus subtilis (B. subtilis) promoter, the vegI promoter (vegIP), has been well studied and shown to be a strong promoter for directing gene expression in gram-positive bacteria. However, when vegIP is harbored in a shuttle vector with a high plasmid copy number (PCN), pM2vegI, it is untransformable in B. subtilis. The plasmid has been found to be transformable in B. subtilis only when a spontaneous point or deletion mutation is introduced at the -11 position of vegIP. My thesis study aims to solve the intransformability issue associated with the use of wild-type vegIP in B. subtilis for the production of recombinant proteins, such as the endoglucanase of Cellulomonas fimi (C. fimi), CenA.

Methodology/Principal Findings: I hypothesize that the intransformability of vegIP harbored by the pM2vegI plasmid may be attributed to the extensive gene transcription promoted by this strong promoter. By co-expressing the lacI repressor, I have demonstrated for the first time that the vegIP-containing plasmid can be transformed in B. subtilis. However, this strategy has been found to be unsuitable for the expression of the reporter gene, cenA, because of plasmid instability. To further minimize the metabolic burden imposed onto the host, vectors with a low PCN are employed instead, and the data show that the lowering of the PCN is a determinant factor for the utilization of vegIP in B. subtilis. Furthermore, I have invented a novel tandem vegIP system (with three consecutive vegIPs) for greatly enhancing the extracellular production of CenA in B. subtilis.

Conclusions/Significance: My thesis research has found that the wild-type vegIP can only be utilized in B. subtilis for directing protein expression, when it is harbored on plasmids with a low PCN. Moreover, vegIP has been demonstrated to be an efficient promoter for heterologous gene expression.