Analysis of the Escherichia coliphoA-based expression systems for production of industrial enzymes

The Escherichia coli phoA gene system, which has a potential to improve the recombinant cell fermentation productivity through its regulatable promoter and signal sequence, was characterized experimentally. An optimal operating scheme to maximize the production of cloned-gene proteins using the phoA gene expression system was developed. The expression of $\beta$-galactosidase from the E. coli phoA-lacZ fusion gene in two different cloning sites (multicopy plasmid and chromosome) revealed that the chromosome integration is more desirable for the tight control of fusion gene expression levels than the insertion into a plasmid. The plasmid-bearing cells showed low degree of overexpression due to a combination of leaky expression in repressed conditions and limitation of biosynthetic machinery in derepressed conditions. In a mixture of inorganic and organic phosphates, inorganic phosphate levels in the medium exhibited oscillatory behavior, so did the cloned protein $\beta$-galactosidase activity. The expression and secretion of Bacillus subtilis $\alpha$-amylase from the phoA-amyE fusion gene in recombinant E. coli under various environmental conditions showed the inhibition of both cell growth and secretion of the native secreted proteins by overexpression of cloned $\alpha$-amylase. The change of culture temperature from 37$\sp\circ$C to 30$\sp\circ$C increased both $\alpha$-amylase and $\beta$-lactamase specific activities by 6 and 2 times, respectively, whereas the AP activity remained unchanged. The increase of stability at post-transcriptional processes (mRNA, translational intermediate, and precursor molecule) and $\alpha$-amylase itself contributed to the enhancement of $\alpha$-amylase activity at 30$\sp\circ$C, while the severe competition for the secretion sites between the secreted proteins seemed to be the main source for the decrease of $\beta$-lactamase activity at 37$\sp\circ$C. The further decrease of the temperature to 25$\sp\circ$C slowed down both cell growth and cloned-gene expression rate. The $\alpha$-amylase activity showed a maximum at pH of 7.4, while AP was most effectively produced at pH of 8.3. A mathematical model was formulated to describe cell growth and product formation in the E. coli phoA-based expression system. The effects of cloning site, product, and culture temperature on cell growth and product formation were compared parametrically. Am optimal operation strategy for the fermentation processes with recombinant E. coli cells containing the phoA-based expression system was developed. For the batch operation, the phosphate-controlled operation showed improved performances over the simple batch operation when total glucose is fixed while the simple batch operation required shorter total operation time than the phosphate-controlled operation