A gene, hmp, which encodes a ubiquitous protein homologous to hemoglobin was isolated among genes from Bacillus subtilis that are induced under anaerobic conditions. The expression of hmp is strongly induced upon oxygen limitation, and the induction is dependent on a two-component regulatory pair, ResD and ResE, an anaerobic regulator, FNR, and respiratory nitrate reductase, NarGHJI.
The objective of this study is to find the optimal Culture system of the hmp promoter to obtain high level of recombinant protein, and characterize the hmp promoter system to test whether this promoter can be used as inducible promoter.
To find the culture conditions for Bacillus subtilis LAB1886, effects of cell density, glucose concentration, pH, DO level and nitrate, nitrite concentration on the cell growth and the expression of β-galactosidase were studied.
From the experiments, the optimal cultivation strategy was found to grow cells in the presence of nitrate to $OD_{600}$ = 0.2 at high DO level (80%), and then to induce the hmp promoter by lowering the DO level to 1-2% with alternating microaerobic and aerobic conditions. Under these conditions, the maximum specific beta-galactosidase activity increased continuously to the maximal value of 1750 Miller units at $OD_{600}$ = 2.5. The best growth medium for culture of B. subtilis LAB1886 among LB, 2YT, and modified R media was 2YT. To maximize induction of the hmp promoter, it was necessary to lower DO levels during induction. In microaerobic condition (DO =1-2%), the expression level of beta-galactosidase was as high as the expression level of beta-galactosidase in completely anaerobic condition. And the cell growth was enhanced in microaerobic condition. In these experiments, accumulation of acetic acid could be kept below 3 g/L. Therefore, acetic acid produced during cultivation might not be a serious problem in this case. However, both of fed-batch and batch cultivation, after the specific beta-galactosidase activity reached to the maximal value, it decreased sharply, and the cell growth represented very low level. To find the reason for these results, effect of nitrite concentration was studied. From the experiments, it was showed that nitrite concentration could affect the expression of beta-galactosidase and the cell growth. When nitrite concentration reached about to 1g/L, the specific beta-galactosidase activity started to decrease, and it reached over to 1.5 g/L, the cell growth was seriously inhibited.
Therefore, nitriteconcentration must be controlled to enhance the cell growth and the expression of β-galactosidase in B. subtilis LAB1886.
