Abstract
High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320), both carrying a heterologous dextransucrase (dsrS) gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction) the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available.High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses.The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the mutated genes in WH320 in addition to the known lacZ.In view of the results of this proteomic study it seems that at high cell density conditions and hence low growth rates MS941, in contrast to WH320, does not maintain a vegetative growth which is essential for the expression of the foreign dsrS gene by using the xylA promoter. It is conceivable that applications of a promoter which is highly active under nutrient-limited cultivation conditions is necessary, at least for MS941, for the overexpression of recombinant genes in such B. megaterium fed-batch cultivation process. However to obtain a heterologous protein in secreted and properly folded form stills remains a big challenge.
Highlights
Bacillus species are frequently used as bacterial workhorses in industrial microbial cultivations for the production of a variety of enzymes as well as fine biochemicals and antibiotics
Characterization of fed-batch cultivations for heterologous dextransucrase production with two recombinant B. megaterium strains The establishment of a high cell density cultivation for B. megaterium and its use for the production of a heterologous dextransucrase (DsrS) from Leuconostoc mesenteroides has been described previously [6]
In this study a further improvement of DsrS yield was achieved with the strain WH320, but significant differences in the production of DsrS were observed between strain WH320 and strain MS941
Summary
Bacillus species are frequently used as bacterial workhorses in industrial microbial cultivations for the production of a variety of enzymes as well as fine biochemicals and antibiotics. The capacity of selected Bacillus strains to produce and secrete large quantities (20–25 g/L) of extracellular enzymes has placed them among the most important industrial enzyme producers. They produce about 60% of the commercially available enzymes [1,2,3,4]. Foreign proteins from different sources have been chosen to explore the capability of B. megaterium as host bacterium for heterologous protein production, such as a dextransucrase (DsrS) from Leuconostoc mensenteroides [6,7], a levansucrase from Lactobacillus reuteri [8], and a hydrolase from Thermobifida fusca [9]. As already evidenced by numerous recent research works it is conceivable that secretion of heterologous proteins can be hampered at different stages of the secretion process, such as poor targeting of heterologous protein precursors to the membrane translocase, resulting in accumulation in the cytoplasm, or limitations in folding and stability of heterologous proteins at the membrane-cell wall interface which render those proteins vulnerable to attacks by wall-associated proteases, or critical interaction of heterologous proteins with the cell wall matrix components, as well as degradation of secreted heterologous proteins by extracellular proteases in the medium
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