The Bacillus megaterium protein production system based on the inducible promoter of the xyl operon (P xylA ) was systematically optimized. Multiple changes in basic promoter elements, such as the ؊10 and ؊35 region and the ribosome-binding site, resulted in an 18-fold increase of protein production compared to the production of the previously established system. The production in shaking-flask culture of green fluorescent protein (Gfp) as a model product led to 82.5 mg per g cell dry weight (g CDW ) or 124 mg liter ؊1 . In fed-batch cultivation, the volumetric protein yield was increased 10-fold to 1.25 g liter ؊1 , corresponding to 36.8 mg protein per g CDW . Furthermore, novel signal peptides for Sec-dependent protein secretion were predicted in silico using the B. megaterium genome. Subsequently, leader peptides of Vpr, NprM, YngK, YocH, and a computationally designed artificial peptide were analyzed experimentally for their potential to facilitate the secretion of the heterologous model protein Thermobifida fusca hydrolase (Tfh). The best extracellular protein production, 5,000 to 6,200 U liter ؊1 (5.3 to 6.6 mg liter ؊1 ), was observed for strains where the Tfh export was facilitated by a codonoptimized leader peptide of YngK and by the signal peptide of YocH. Further increases in extracellular protein production were achieved when leader peptides were used in combination with the optimized expression system. In this case, the greatest extracellular enzyme amount of 7,200 U liter ؊1 , 7.7 mg liter ؊1 , was achieved by YocH leader peptide-mediated protein export. Nevertheless, the observed principal limitations in protein export might be related to components of the Sec-dependent protein transport system.
A multiple vector system for the production and export of recombinant affinity-tagged proteins in Bacillus megaterium was developed. Up to 1 mg/liter of a His 6 -tagged or Strep-tagged Lactobacillus reuteri levansucrase was directed into the growth medium, using the B. megaterium esterase LipA signal peptide, and recovered by one-step affinity chromatography.The gram-positive bacterium Bacillus megaterium has several advantages over other recombinant protein production hosts. In contrast to Bacillus subtilis, B. megaterium does not possess alkaline proteases and is known for the stable replication and maintenance of plasmids (7). The bacterium readily secretes proteins into the growth medium. For this study, the commercially interesting levansucrase Lev from Lactobacillus reuteri 121 (3, 6) was chosen as a model protein to further improve the efficiency and use of a novel B. megaterium-based secretion system for heterologous proteins.
Gene expression systems based on the RNA polymerase of the bacteriophage T7 are often the ultimate choice for the high level production of recombinant proteins. During the last decade, the Gram-positive bacterium Bacillus megaterium was established as a useful host for the intra- and extracellular production of heterologous proteins. In this paper, we report on the development of a T7 RNA polymerase-dependent expression system for B. megaterium. The system was evaluated for cytosolic and secretory protein production with green fluorescent protein (GFP) from Aequoria victoria as intracellular and Lactobacillus reuteri levansucrase as extracellular model protein. GFP accumulated rapidly at high levels up to 50 mg/l shake flask culture intracellularly after induction of T7 RNA polymerase gene expression. The addition of rifampicin for the inhibition of B. megaterium RNA polymerase led to an increased stability of GFP. L. reuteri levansucrase was also successfully produced and secreted (up to 20 U/l) into the culture supernatant. However, parallel intracellular accumulation of the protein indicated limitations affiliated with the Sec-dependent protein translocation process.
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