In this work, the protein production potential of the xylose-inducible expression system for B. megaterium was systematically improved. Enhanced transcription was achieved by the insertion of consensus promoter elements for the −10 or −35 region. mRNA was improved by modifications which led to a predicted hairpin loop in the 5′ untranslated region of the target gene's mRNA. Furthermore, a ribosome binding site (rbs) adapted to B. megaterium 16S rRNA was applied to accelerate translation. Employed individually, all of these genetic elements yielded positive effects on recombinant protein production in B. megaterium. The combinations of −10+ and rbs+ or utr+ and rbs+ facilitated strong improvements in Gfp formation (62.9 mg per gCDW and 75.0 mg per gCDW). Surprisingly, these levels of production were much greater than the sums of the positive effects of these elements when tested individually. The resulting increases in protein production were nearly multiplications of the fold changes observed for the single optimizations. Obviously, a larger amount or a more stable mRNA represents an improved target for the ribosome, which is attracted by the perfect Shine-Dalgarno sequence. Taken together, this yields a highly synergistic protein synthesis process.
The optimization of both the −35 and −10 region, resulting in a complete consensus promoter for gene expression, was realized in pSSBm81 (−35+
). Surprisingly, this led to lower protein yields (16.7 mg per gCDW
) and the loss of external promoter control. Ellinger et al. (11
) observed RNA polymerase stalling at promoters with very high similarity to the consensus during in vivo
experiments in E. coli
. Obviously hampered by strong promoter affinity, the rate-limiting step was found to be the release of the sigma factor and, thus, the transition to an elongation complex (11
). Here, this stalling effect might have led to reduced transcription rates which consequently resulted in the formation of smaller amounts of Gfp. Under noninducing conditions, detectable amounts of Gfp were found, indicating a loss of promoter controllability (data not shown). Due to the highly increased affinity of the RNA polymerase complex to the consensus promoter (−35+
), the XylR repressor binding might have lost its competitiveness. Since the binding sequence for the XylR repressor is located just a few base pairs downstream of PxylA
, it seems probable that the major repressing effect of XylR is caused by competitive binding to the promoter region, as was shown for the lac
repressor of E. coli
by Schlax et al. (25
). B. megaterium
cells producing Gfp mediated by a plasmid containing a combination of −35+
, and rbs+
showed behavior similar to that of cells harboring pSSBm81 (−35+
) (data not shown). This might indicate that DNA sequence changes introduced to utr+
do not influence the mRNA's half-life exclusively but also significantly increase the RNA polymerase-binding affinity to its promoter.
B. megaterium cells employing pSSBm78 (−35+ rbs+) produced only a little more Gfp (80.6 mg per gCDW) than cells carrying pSSBm84 (−35+) (76.3 mg per gCDW). Here, the expected multiplier effect was not detected. Although the introduction of the NheI restriction site into the nonoptimized basic vector p3STOP1623 caused a significant reduction in Gfp production, almost no differences in Gfp production were measured when comparing cells carrying the optimized plasmids with or without the NheI restriction site (pSSBm78, 80.6 mg per gCDW; pSSBm85, 82.5 mg per gCDW). These observations led to speculation about general limitations of the protein formation process. On one site, this might be due to specific characteristics of the target gene. For example, the augmented use of certain codons that are rare in B. megaterium might lead to limitation of the corresponding tRNAs and would therefore hamper protein production. Another reason might be the limited supply of specific amino acids. This theory is supported by observations made during the fed-batch cultivations. Here, on the one hand, high volumetric Gfp yields (1.25 g liter−1) were achieved, but on the other hand, only smaller amounts of Gfp per unit of cell dry weight (36.8 mg per gCDW) than in shaking-flask cultures (82.5 mg per gCDW) were reached. An explanation might be the use of defined synthetic medium in the fed-batch cultivations and of complex medium in the shaking flasks.
Nevertheless, the combined optimized features employed in the new high-performance expression plasmids facilitated the production of 82.5 mg per gCDW
Gfp in B. megaterium
shaking-flask cultivations, which is even more than described for fed-batch cultivations of E. coli
(73.7 mg per gCDW
). Thus, a competitive protein production system for the alternative bacterial host B. megaterium
In experiments in the production and secretion of heterologous Tfh in B. megaterium, the secretion facilitated by the mediocre SPPac was enhanced up to 6-fold when employing the optimized high-performance plasmids. However, the amount of Tfh secreted was not strongly enhanced when using well-suited SPs like SPLipA, SPYocH, or SP+YngK. Neither the new signal peptides that were tested nor their use in combination with the high-performance plasmids facilitated more than 16% greater secretion of Tfh (SPYocH in high-performance plasmid, 7,200 U liter−1, which equals 7.7 mg liter−1) than was mediated by SPLipA used in combination with the nonoptimized expression system (6,200 U liter−1 or 6.6 mg liter−1). These results clearly show limitations of the Sec-dependant secretion process, since the intracellular protein production was drastically increased, up to 18-fold (4.6 versus 82.5 mg per gCDW), as shown for Gfp production employing the high-performance production system.
Furthermore, it was shown that a computational approach to create an artificial signal peptide is in part possible. The application of an SPAsp
-containing expression plasmid in protein secretion experiments proved the sufficient biological functionality of the artificial leader peptide. Since no prediction method for the right combination of SP and target protein is currently applicable with success (6
), screening for the correct SP-protein combination is still necessary. Nevertheless, in combination with the newly developed high-performance expression system, six (SPYocH
, and SPLipA
) out of eight SPs tested (75%) facilitated high-yield secretion of Tfh, while only three SPs (SPYocH
, and SPLipA
) (38%) achieved an equivalent performance when employed in the nonoptimized production system. The described toolbox comprising high-performance expression plasmids for both intracellular and extracellular protein production is commercially available from MoBiTec GmbH, Göttingen, Germany.