초록
<P><B>Background</B></P><P><I>Bacillus subtilis</I> is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in <I>B. subtilis</I> 168 cells overproducing the industrially relevant α-amylase AmyM from <I>Geobacillus stearothermophilus</I>, which was expressed from the strong promoter <I>P(amyQ)</I>-<I>M</I>.</P><P><B>Results</B></P><P>Here we show that activity of the <I>htrB</I> promoter as induced by overproduction of AmyM was “noisy”, which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of <I>P(amyQ)</I>-<I>M</I> promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with <I>gfpmut3.</I> Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of <I>P(amyQ)</I>-<I>M</I>. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to <I>P(amyQ)</I>-<I>M</I> activity were observed for the <I>B. subtilis</I> mutant strain 168<I>degUhy3</I>2, and the wild-type strain 168 under optimized growth conditions.</P><P><B>Conclusion</B></P><P>Expression heterogeneity of secretory proteins in <I>B. subtilis</I> can be suppressed by <I>degU</I> mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and <I>gfp</I> represents a versatile tool for real-time monitoring of protein production and opens novel avenues for <I>Bacillus</I> production strain improvement.</P>