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Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase.

Wang W, Hollmann R, Deckwer WD - Proteome Sci (2006)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemical Engineering, Technical University Braunschweig, GBF/TU-BCE, Mascheroder Weg 1, D-38124 Braunschweig, Germany. wwa@gbf.de

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.

No MeSH data available.


Related in: MedlinePlus

Sections of 2-DE PAGE gel images for the visualization of expression changes of some proteins. DnaK: heat shock 70 kDa chaperone DnaK; GroEL: 60 kDa chaperonin GroEL; EF-G: elongation factor G; TIG: trigger factor; TKT: transketolase; Bmg4247 and Bmg0510: peptidoglycan-binding proteins; Bmg4479: HtrA-type membrane-bound quality control protease; AcoB: acetoin dehydrogenase E1 component.
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Figure 5: Sections of 2-DE PAGE gel images for the visualization of expression changes of some proteins. DnaK: heat shock 70 kDa chaperone DnaK; GroEL: 60 kDa chaperonin GroEL; EF-G: elongation factor G; TIG: trigger factor; TKT: transketolase; Bmg4247 and Bmg0510: peptidoglycan-binding proteins; Bmg4479: HtrA-type membrane-bound quality control protease; AcoB: acetoin dehydrogenase E1 component.

Mentions: Pyruvate kinase (PYK) demonstrated a nearly constant expression level for WH320 during the whole sampling time period but a clearly reduced expression level in the fed-batch phase for MS941 (Figure 4). Similar to PYK expression profiles all the subunits of the downstream pyruvate dehydrogenase complex (PdhA, PdhB, PdhC and PdhD), were clearly down-regulated in the fed-batch phase for MS941, whereas most of them remained relatively constant for WH320 (Figure 4). Furthermore, differences were also observed between the two strains in the expression patterns of some enzymes involved in the pentose phosphate pathway. For instance, transketolase (TKT), a key enzyme involved in the non-oxidative PP pathway to create a bridge between glycolysis and the PP pathway, showed expression patterns similar to PYK and PDH complex, namely relatively constant expression in WH320 but reduced expression in MS941, as can be visualized on the sections of the 2-D PAGE gel images (Figure 5).


Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase.

Wang W, Hollmann R, Deckwer WD - Proteome Sci (2006)

Sections of 2-DE PAGE gel images for the visualization of expression changes of some proteins. DnaK: heat shock 70 kDa chaperone DnaK; GroEL: 60 kDa chaperonin GroEL; EF-G: elongation factor G; TIG: trigger factor; TKT: transketolase; Bmg4247 and Bmg0510: peptidoglycan-binding proteins; Bmg4479: HtrA-type membrane-bound quality control protease; AcoB: acetoin dehydrogenase E1 component.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1622742&req=5

Figure 5: Sections of 2-DE PAGE gel images for the visualization of expression changes of some proteins. DnaK: heat shock 70 kDa chaperone DnaK; GroEL: 60 kDa chaperonin GroEL; EF-G: elongation factor G; TIG: trigger factor; TKT: transketolase; Bmg4247 and Bmg0510: peptidoglycan-binding proteins; Bmg4479: HtrA-type membrane-bound quality control protease; AcoB: acetoin dehydrogenase E1 component.
Mentions: Pyruvate kinase (PYK) demonstrated a nearly constant expression level for WH320 during the whole sampling time period but a clearly reduced expression level in the fed-batch phase for MS941 (Figure 4). Similar to PYK expression profiles all the subunits of the downstream pyruvate dehydrogenase complex (PdhA, PdhB, PdhC and PdhD), were clearly down-regulated in the fed-batch phase for MS941, whereas most of them remained relatively constant for WH320 (Figure 4). Furthermore, differences were also observed between the two strains in the expression patterns of some enzymes involved in the pentose phosphate pathway. For instance, transketolase (TKT), a key enzyme involved in the non-oxidative PP pathway to create a bridge between glycolysis and the PP pathway, showed expression patterns similar to PYK and PDH complex, namely relatively constant expression in WH320 but reduced expression in MS941, as can be visualized on the sections of the 2-D PAGE gel images (Figure 5).

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemical Engineering, Technical University Braunschweig, GBF/TU-BCE, Mascheroder Weg 1, D-38124 Braunschweig, Germany. wwa@gbf.de

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.

No MeSH data available.


Related in: MedlinePlus