Engineering Bacillus megaterium for production of functional intracellular materials

Abstract

Background: 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. 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 bioengineered 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 PHB production in 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.

Keywords: Bacillus megaterium; Endotoxin; Functionalized beads; Genetic engineering; IgG binding; PHA synthase; Poly(3-hydroxybutyrate) (PHB); Sporulation; ZZ-domain; ∆spoIIE.

Fig. 1

Transmission electron microscopic analysis of B. megaterium PHA05 cells containing recombinantly produced PHB beads. The ZZ-domain displaying beads were produced from plasmid pPT7-ZZCAB

Fig. 2

SDS-PAGE of ZZ-displaying and wild type beads recombinantly produced in B. megaterium PHA05 under control of the T7 promoter. Lane 1, ZZ-displaying beads, ZZ/ZZ-PhaC-ZZ protein at one asterisk (expected molecular weight 118 kDa); lane 2, wild type beads, PhaC protein at two asterisks (expected molecular weight 118 kDa). Equal amounts of protein as determined by Bradford were loaded in each lane. L, Mark 12™ protein standard (Thermo Fisher Scientific). The identity of the ZZ/ZZ-PhaC-ZZ fusion protein was confirmed by MALDI-TOF(MS)

Fig. 3

IgG binding assays using a purified IgG and b human serum. F, feed [IgG (a) or human serum (b)]; U unbound, E elution, CAB control beads not displaying ZZ domain, ZZ ZZ-domain displaying beads produced in this study, GE commercially available GE protein A beads; L, Mark 12™ protein standard (Thermo Fisher Scientific). Beads were incubated with the respective feed material, washed repeatedly and IgG eluted with glycine pH 2.7. Asterisks indicate the IgG light and heavy chains

Fig. 4

a Overview of spoIIE disruption by single crossover (Campbell-like integration) and b confirmation of spoIIE disruption by PCR amplification. a The constructed plasmid unable to replicate in the host organism is depicted. It contains a section of the target gene (between FRT sites) plus a selectable marker (Cm^R). Homologous recombination occurs between the gene fragment on the plasmid and the identical gene segment on the chromosome followed by genome integration of the entire plasmid. The target gene is ‘insertionally inactivated’, i.e. disrupted by splitting it into a truncated forward and rear end (with the homologous gene sequence present twice in the altered genome). b Amplification across each junction was done using one primer binding in the genome and one primer binding within the integrated plasmid-derived fragment. 5′ a, amplification across 5′ junction using primers spo5′fwd2 and CmF (1.9 kb product); 5′ b, amplification across 5′ junction using primers spo5′fwd3 and Cm_pMK_inner_fwd2 (1.2 kb product); 3′, amplification across 3′ junction using primers M13 fwd and spo3′rev2 (2.3 kb product); ΔE, spoIIE knockout strain; C, B. megaterium PHA05 wild type control; P, plasmid control (pFRT-Cm-spoIIE). For supplemental confirmation, the PCR product obtained with primers spo5′-fwd2 and CmF was sequenced. L, ladder λ PstI (PstI digested λ DNA)