Physiological and Genetic Strategies for Enhanced Subtilisin Production by Bacillus subtilis

Abstract

Defined minimal media conditions were used to assess and subsequently enhance the production of subtilisin by genetically characterized Bacillus subtilis strains. Subtilisin production was initiated by the exhaustion or limitation of ammonium in batch and fed-batch cultures. Expression of the subtilisin gene (aprE) was monitored with a chromosomal aprE::lacZ gene fusion. The beta-galactosidase production driven by this fusion reflected subtilisin accumulation in the culture medium. Subtilisin gene expression was temporally extended in sporulation-deficient strains (spoIIG), relative to co-genic sporogenous strains, resulting in enhanced subtilisin production. Ammonium exhaustion not only triggered subtilisin production in asporogenous spoIIG mutants but also shifted carbon metabolism from acetate production to acetate uptake and resulted in the formation of multiple septa in a significant fraction of the cell population. Fed-batch culture techniques, employing the spoIIG strain, were investigated as a means to further extend subtilisin production. The constant provision of ammonium resulted in linear growth, with doubling times of 11 and 36 h in each of two independent experiments. At the lower growth rate, the responses elicited (subtilisin production, glucose metabolism, and morphological changes) during the feeding regime closely approximated the ammonium starvation response, while at the higher growth rate a partial starvation response was observed.

Amplification of the Escherichia coli lacZ gene in Bacillus subtilis and its expression on a by‐product growth medium

“…However, at late stationary phase, the specific activity values recorded by the mutant strain were markedly higher than those achieved by the wild type culture. Similar results have been obtained when a spoIIG oligosporogenous B. subtilis mutant was used for alkaline protease production (PIERCE et al 1992).…”

“…Resistance amplification to a sublethal antibiotic dose of 140 µg/ml resulted in about 3.8-fold enzyme over-expression. The obtained specific level of E-galactosidase, calculated in U/mg protein (17.8 K 10 3 ), is approximately 8-and 2.5-fold higher than the maximum levels reported by PIERCE et al (1992) andPARK et al (1992), respectively.…”

Morphological and biochemical responses of Bacillus subtilis to selenite stress

“…subtilis 168 (prototrophic strain) was grown in a minimal chemically defined liquid MOPS medium [21] [8]. subtilis 168 (prototrophic strain) was grown in a minimal chemically defined liquid MOPS medium [21] [8].…”

“…B. subtilis 168 (prototrophic strain) was grown in a minimal chemically defined liquid MOPS medium [21] [8]. The medium was supplemented with 1 mM sodium selenite.…”

New Integrative Method To Generate Bacillus subtilis Recombinant Strains Free of Selection Markers

“…In the second case, selection of the strain which has lost resistance is tedious due to the relatively low frequencies and absence of positive selection. In the same way, the optimization of a recombinant B. subtilis strain for overproduction and secretion of a protein can require chromosomal modifications which could involve the integration of several copies of the gene of interest (7), the construction of multiprotease-and sporulation-deficient strains (18,19,25,26), and/or the coexpression of chaperones to amplify the posttranscriptional maturation of the protein (2,24). Another example of the usefulness of generating multiple mutants is for the study of complex physiological pathways, such as sporulation.…”