RESUMEN
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 codon-optimized 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.