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

BACKGROUND: Bacillus subtilis 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 B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M. RESULTS: Here we show that activity of the htrB 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 P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions. CONCLUSION: Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement.

APHO1 from the yeast Arxula adeninivorans encodes an acid phosphatase of broad substrate specificity.

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36-39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60 degrees C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.