11/13/2023 0 Comments Bacillus megaterium spore stainThe last PHB synthesis reaction is catalysed by the PHB Synthase (PhaC) which remains covalently bound to the emerging PHB granule. PHB is synthesized from the central metabolite acetyl-CoA, requiring consecutive conversions catalysed by three enzymes. Polyhydroxyalkanoates (PHAs) in general serve as carbon and energy storage. Bacterial inclusions made of the biopolyester, poly(3-hydroxybutyrate) (PHB), have been extensively redesigned using bioengineering and synthetic biology approaches in order to obtain surface functionalized nano-/micro-spheres. This sporulation-negative mutant represents an improved industrial production strain for biotechnological processes otherwise impaired by the possibility of endospore formation.īacteria have been engineered to produce protein or polymer inclusions in order to efficiently recover proteins of interest or to immobilize and/or display various protein functions for medical and industrial uses. megaterium incapable of sporulation but particularly suitable for production of functionalized PHB beads. Disruption of the spoIIE gene rendered B. megaterium as a host for the production of functionalized PHB beads. This study provides proof of concept for the successful genetic engineering of B. This strain did not produce spores when tested under sporulation inducing conditions and it was still able to synthesize ZZ-domain displaying PHB beads. megaterium has the ability to sporulate and respective endospores could co-purify with cellular inclusions, a sporulation negative production strain was generated by disrupting the spoIIE gene in PHA05. The ZZ-domain PhaC fusion protein was strongly overproduced at the surface of the PHB inclusions and the corresponding isolated ZZ-domain displaying PHB beads were found to purify IgG with a binding capacity of 40–50 mg IgG/g beads. To produce functionalized PHB inclusions, the N- and C-terminus of PhaC was fused to four and two IgG binding Z-domains from Staphylococcus aureus, respectively. Plasmid-mediated T7 promoter-driven expression of the genes encoding β-ketothiolase ( phaA), acetoacetyl-CoA-reductase ( phaB) and PHB synthase ( phaC) enabled PHB production in B. megaterium is a natural PHB producer, the PHB-negative strain PHA05 was used to avoid any background PHB production. In this study, we have bioengineered the formation of functional PHB inclusions in the Gram-positive bacterium Bacillus megaterium, an LPS-free and established industrial production host. So far, beads have mainly been produced in recombinant Escherichia coli, which is problematic for some applications as the lipopolysaccharides (LPS) co-purified with such inclusions are toxic to humans and animals. The respective fusion protein mediates self-assembly of PHB inclusions displaying the desired protein function. This has been achieved by translational fusion of foreign proteins to the PHB synthase (PhaC). Over the last 10–15 years, a technology has been developed to engineer bacterial poly(3-hydroxybutyrate) (PHB) inclusions as functionalized beads, for applications such as vaccines, diagnostics and enzyme immobilization.
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