Genome integration and expression of β-glucosidase in Priestia megaterium enhanced poly(3-hydroxybutyrate) production from cellobiose and cellulose

Polyhydroxyalkanoates (PHAs) production using cellulosic biomass is a promising way for sustainable manufacturing of bioplastics. Priestia megaterium is an ideal choice as it can use glucose and xylose for PHA production. To further improve the strain for PHA production from cellobiose, we integrate exogenous β-glucosidase (Bgl) from Bacillus sp. GL1 (Bsbgl) in the PHA depolymerase (phaZ1) deletion background (ΔZ1) using CRISPR-Cas. The deletion of phaZ1 in P. megaterium showed a significant improvement in the PHA accumulation whereas BsBgl expression resulted in robust activity and improved growth using cellobiose as a sole carbon source compared to other Bgl targets. To further improve the strain, four native promoters were examined for intracellular BsBgl expression, and the PHA promoter (P_phaR) and citrate synthase promoter (P_citz) showed 2.0- and 4.5-fold higher activities of BsBgl, compared to the xylose promoter (P_xyl). The rate of cellobiose utilization in engineered strains P2 (P_phaRBsbgl_ΔZ1) and P4 (P_citzBsbgl_ΔZ1) was improved to 1.6-fold and 2.6-fold, whereas poly(3-hydroxybutyrate) (P3HB) yield for the respective strains was around 3-fold to the wild-type. The strain P2 turned out to be better for cellobiose to PHA production. Further, the strain P2 in a co-culture with cellulolytic Streptomyces sp. SirexAA-E in a consolidated bioprocessing yielded 76 mg of P3HB/ g of carboxymethylcellulose, which is 4.3-times higher than the co-culture with the wild-type. Thus, the present work improved the cellobiose utilization and P3HB accumulation of P. megaterium. The current study paves the way for designing efficient cell factories for cellulosic biomass into bioplastic in the future.