Supplementary Material for: Role of <i>CcpA</i> in Polyhydroxybutyrate Biosynthesis in a Newly Isolated <i>Bacillus</i> sp. MA3.3

The <i>ccpA</i> gene was inactivated in the polyhydroxybutyrate (PHB)-producing strain <i>Bacillus</i> sp. MA3.3 in order to reduce glucose catabolite repression over pentoses and develop improved bacterial strains for the production of PHB from lignocellulosic hydrolysates. Mutant <i>Bacillus</i> sp. MSL7 ΔCcpA are unable to grow on glucose and ammonia as sole carbon and nitrogen sources, respectively. Supplementation of glutamate as the nitrogen source or the substitution of the carbon source by xylose allowed the mutant to partially recover its growth performance. RT-PCR showed that CcpA stimulates the expression of the operon (<i>gltAB)</i>,responsible for ammonia assimilation via glutamate in <i>Bacillus </i>sp. MA3.3. Moreover, it was demonstrated that the supplementation of xylose or glutamate was capable of stimulating <i>gltAB</i> operon expression independently of CcpA. In PHB production experiments in mineral media, it has been observed that the glucose catabolite repression over the pentoses was partially released in MSL7. Although the carbohydrate consumption is faster in the <i>ccpA</i> mutant, the biomass and PHB biosynthesis are lower, even with supplementation of glutamate. This is attributed to an increase of acetyl-CoA flux towards the tricarboxylic acid cycle observed in the mutant.