The "beauty in the beast"-the multiple uses of Priestia megaterium in biotechnology

Abstract

Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B₁₂, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B₁₂ (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. KEY POINTS: • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.

Keywords: Bacillus megaterium; Cell-free transcription-translation; Cytochrome P450; Plant growth-promoting bacterium; Polyhydroxybutyrate (PHB); Priestia megaterium; Recombinant protein production; Vitamin B12.

Fig. 1

Electron microscope image of Priestia megaterium (large cells) and Escherichia coli (small cells). P. megaterium and E. coli were individually grown aerobically in rich medium at 37 °C, mixed in the middle of their exponential growth phases and examined in a field emission scanning electron microscope (FESEM) Zeiss DSM982 Gemini (magnification 6,500-fold). The picture was taken by Manfred Rohde, Helmholtz Centre for Infection Research, Braunschweig, Germany.

Fig. 2

Summary of cobalamin genetics, biosynthesis, and regulation in Priestia megaterium DSM319. Upper part: cobalt and cobalamin transporters are indicated in yellow, cobalt in pink, and cobalt chaperon in green. Middle part: summary of cobalamin biosynthesis starting from 8 molecules of 5-aminolevulinic acid. The final product here is shown as adenosylcobalamin which can interact with the cobalamin riboswitches. CobI and CobII indicate all enzymes encoded by the cobI and cobII operons shown below. Lower part: all genes are represented as colored arrows. Black arrows upstream of the operons/single genes indicate promoters, black “T”s terminators and black stem-loop structures cobalamin riboswitches. All genes clustered in operons or situated on their own are annotated. Hypothetical genes are annotated as open reading frames (bmd_0000) as shown in www.megabac.tu-bs.de.

Fig. 3

Schematic summary of Priestia megaterium plasmids used for the production, secretion, and purification of recombinant proteins. All plasmids are constructed as shuttle plasmids for cloning in E. coli (yellow elements) and replication (dark blue, different compatibility classes), selection (blue), and production of recombinant proteins in P. megaterium. Suitable promoters (black arrow) are the native (P_xylA) and the optimized (P_xylA^opt.) xylose-inducible promoter, the lactose inducible (P_lac), sucrose (P_suc, P_sacB), arabinose (P_ara), galactosidase (P_gal), IPTG (P_Hysp), and starch (P_amyL) promoter, the T7-RNA-polymerase-dependent promoter which is based on a two-plasmid system, and several constitutive (P_const) and growth phase-dependent (P_growth) promoters. Genes encoding recombinant proteins can be fused to coding sequences of different signal peptides (purple) of the lipase A (SP_LipA), the unknown secreted proteins YocH (SP_YocH) and YngK (SP_YngK), the natural protease NprM (SP_NprM), and the serine protease VPR (SP_Vpr). In addition original signal peptides of the foreign recombinant protein can be used (SP_native). For purification of intra- or extracellular recombinant proteins, a fusion to N- or C-terminal His₆ or StrepII tag is possible (orange). N-terminal tags can be removed of using tobacco each virus (TEV) or factor X_a protease cleavage (light blue). Black stars indicate stable places for integration of additional genetic elements as tRNAs or genes for co-expression.