Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions

• DOI: 10.1186/s12934-016-0455-1
• Publication Date: 2016-03-29T15:17:21Z
• Authors: Tina N. Ploss; Ewoud Reilman; Carmine G. Montef...; Emma L. Denham; Sjouke Piersma; Anja Lingner; Jari Vehmaanperä; Patrick Lorenz; Jan Maarten van Dijl
• Keywords: Overproduction; Secretory protein

Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability secrete commercially relevant proteins in large amounts directly into growth medium. However, hyper-secretion of proteins, such as α-amylases, leads induction secretion stress-responsive CssR-CssS regulatory system, resulting up-regulation HtrA and HtrB proteases. These proteases degrade misfolded secreted via Sec pathway, a loss product. The aim this study was investigate stress response B. 168 cells overproducing industrially α-amylase AmyM from Geobacillus stearothermophilus, which expressed strong promoter P(amyQ)-M. Here we show that activity htrB induced by overproduction “noisy”, indicative heterogeneous activation pathway. Plasmids were constructed allow real-time analysis P(amyQ)-M production by, respectively, transcriptional out-of-frame translationally coupled fusions with gfpmut3. Our results emergence distinct sub-populations high- low-level AmyM-producing cells, reflecting heterogeneity This most likely explains response. Importantly, more homogenous populations regard observed mutant strain 168degUhy32, wild-type under optimized conditions. Expression secretory can be suppressed degU mutation Further, translational fusion gene target protein gfp represents versatile tool monitoring opens novel avenues improvement.