Construction of self-disruptive Bacillus megaterium in response to substrate exhaustion for polyhydroxybutyrate production

Abstract.

In order to establish a novel recovery system for polyhydroxyalkanoates, a self-disruptive strain of Bacillus megaterium that responds to substrate exhaustion was constructed. A gene cassette carrying the lysis system of Bacillus amyloliquefaciens phage – holin and endolysin – was inserted into the Escherichia coli-Bacillus subtilis shuttle vector pX under the control of a xylose-inducible expression system, xylR-xylA ′. In this system, the expression of a target gene is induced by xylose but inhibited by glucose, which acts as an anti-inducer. B. megaterium was transformed with pX conveying the phage lysis system, which was integrated into the amyE locus of chromosomal DNA of B. megaterium by homologous recombination. The lysis system caused self-disruption of the transformant cells effectively even when expression of the lysis genes was induced during stationary phase. For the production of polyhydroxybutyrate (PHB), the transformant was grown in a medium containing glucose as a substrate in the presence of xylose. When the glucose concentration approached zero, self-disruption was spontaneously induced, releasing intracellularly accumulated PHB into the culture broth. This system realizes timely cell disruption immediately after the PHB content in the cell reaches a maximum level.