Engineering Bacillus megaterium for production of functional intracellular materials

Simple item page
dc.citation.volume16
dc.contributor.authorGrage K
dc.contributor.authorMcDermott P
dc.contributor.authorRehm BHA
dc.date.available22/11/2017
dc.date.issued22/11/2017
dc.description.abstractBackground: Over the last 10-15 years, a technology has been developed to engineer bacterial polyhydroxybutyrate (PHB) inclusions as functionalized beads, for applications such as vaccines, diagnostics and enzyme immobilization. This has been achieved by translational fusion of foreign proteins to the PHB synthase (PhaC). The respective fusion protein mediates self-assembly of PHB inclusions displaying the desired protein function. 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. Results: In this study, we have engineered the formation of functional PHB inclusions in the Gram-positive bacterium Bacillus megaterium, an LPS-free and established industrial production host. As B. megaterium is a natural PHB producer, the PHB-negative strain PHA05 was used to avoid any background PHB production. Plasmid-mediated T7 promoter-driven expression of the genes encoding β-ketothiolase (phaA), acetoacetyl-CoA-reductase (phaB) and PHB synthase (phaC) enabled/effected PHB production by B. megaterium PHA05. 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. 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. As B. 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. This strain did not produce spores when tested under sporulation inducing conditions and it was still able to synthesize ZZ-domain displaying PHB beads. Conclusions: This study provides proof of concept for the successful genetic engineering of B. megaterium as a host for the production of functionalized PHB beads. Disruption of the spoIIE gene rendered B. megaterium incapable of sporulation but particularly suitable for production of functionalized PHB beads. This sporulation-negative mutant represents an improved industrial production strain for biotechnological processes otherwise impaired by the possibility of endospore formation.
dc.description.confidentialFALSE
dc.identifierhttps://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-017-0823-5
dc.identifier.citationMicrobial Cell Factories, 2017, 16
dc.identifier.doi10.1186/s12934-017-0823-5
dc.identifier.elements-id387076
dc.identifier.harvestedMassey_Dark
dc.identifier.issn1475-2859
dc.identifier.urihttps://hdl.handle.net/10179/13284
dc.publisherBioMed Central
dc.publisher.urihttps://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-017-0823-5
dc.relation.isPartOfMicrobial Cell Factories
dc.subjectPolyhydroxybutyrate (PHB), PHA synthase, Bacillus megaterium, endotoxin, genetic engineering, functionalized beads, ZZ domain, IgG binding, sporulation, ∆spoIIE
dc.subject.anzsrc0605 Microbiology
dc.subject.anzsrc1003 Industrial Biotechnology
dc.titleEngineering Bacillus megaterium for production of functional intracellular materials
dc.typeJournal article
pubs.notesNot known
pubs.organisational-group/Massey University
pubs.organisational-group/Massey University/College of Sciences
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Grage_et_al-2017-Microbial_Cell_Factories.pdf
Size:
1.47 MB
Format:
Adobe Portable Document Format
Description:
Download
Collections
Journal Articles