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Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases.

Klinger J, Fischer R, Commandeur U - Front Plant Sci (2015)

Bottom Line: Only the β-glucosidase showed high activity against 4-MUC.In contrast, all the plant-derived enzymes were active against the respective model substrates.Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biology VII (Molecular Biotechnology), RWTH Aachen University Aachen, Germany.

ABSTRACT
The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

No MeSH data available.


Related in: MedlinePlus

Western blot and Coomassie stained gel of Escherichia coli cell extracts and cell pellet fractions. Proteins were detected with monoclonal anti-polyhistidine antibodies. His-tagged mCherry was used as a positive control (+) and a cell extract from E. coli transformed with an empty vector (-) was used as a negative control. Lanes for BglC and E1–E6 contain cell extracts from cultures transformed with the corresponding pJK vectors for cellulase expression. Per lane, 5 μg total soluble protein (TSP) (A) or 15 μg TSP (B) were loaded. (A) Depicts a blot of the cell lysate with the corresponding Coomassie stained gel. (B) Depicts a blot of the cell pellet fraction with the corresponding Coomassie stained gel.
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Figure 2: Western blot and Coomassie stained gel of Escherichia coli cell extracts and cell pellet fractions. Proteins were detected with monoclonal anti-polyhistidine antibodies. His-tagged mCherry was used as a positive control (+) and a cell extract from E. coli transformed with an empty vector (-) was used as a negative control. Lanes for BglC and E1–E6 contain cell extracts from cultures transformed with the corresponding pJK vectors for cellulase expression. Per lane, 5 μg total soluble protein (TSP) (A) or 15 μg TSP (B) were loaded. (A) Depicts a blot of the cell lysate with the corresponding Coomassie stained gel. (B) Depicts a blot of the cell pellet fraction with the corresponding Coomassie stained gel.

Mentions: Samples were harvested from E. coli cultures and N. tabacum leaves and the resulting extracts were used directly for western blotting and activity assays. To detect possible negative effects on E. coli growth behavior related to cellulase expression, OD600 at the end of the cultivation time was measured and compared to the empty vector control. Table 2 shows the average OD600 measured from four independent cultivations. This showed no clear negative effect of the cellulase expression on the final cell density. The bacterial extracts yielded bands in the western blot indicating the successful expression of all genes except those encoding exocellulases E3 and E6 (Figure 2A). The mobility of each band was consistent with the theoretical mass of the corresponding protein (Table 3), showing the successful expression of full-length proteins. Furthermore, each construct produced a single band on the blot suggesting the absence of protein degradation products. The blot for the cell pellet fraction (Figure 2B) showed bands only for BglC, E1, E4, and E5 indicating that a part of the protein remains insoluble. An additional band was observed for E1 indicating a slight degradation or a potential isoform of the enzyme when present in the insoluble fraction. For the exocellulases E3 and E6 as well as the endocellulase E2 there were no bands observed.


Comparison of Thermobifida fusca Cellulases Expressed in Escherichia coli and Nicotiana tabacum Indicates Advantages of the Plant System for the Expression of Bacterial Cellulases.

Klinger J, Fischer R, Commandeur U - Front Plant Sci (2015)

Western blot and Coomassie stained gel of Escherichia coli cell extracts and cell pellet fractions. Proteins were detected with monoclonal anti-polyhistidine antibodies. His-tagged mCherry was used as a positive control (+) and a cell extract from E. coli transformed with an empty vector (-) was used as a negative control. Lanes for BglC and E1–E6 contain cell extracts from cultures transformed with the corresponding pJK vectors for cellulase expression. Per lane, 5 μg total soluble protein (TSP) (A) or 15 μg TSP (B) were loaded. (A) Depicts a blot of the cell lysate with the corresponding Coomassie stained gel. (B) Depicts a blot of the cell pellet fraction with the corresponding Coomassie stained gel.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664618&req=5

Figure 2: Western blot and Coomassie stained gel of Escherichia coli cell extracts and cell pellet fractions. Proteins were detected with monoclonal anti-polyhistidine antibodies. His-tagged mCherry was used as a positive control (+) and a cell extract from E. coli transformed with an empty vector (-) was used as a negative control. Lanes for BglC and E1–E6 contain cell extracts from cultures transformed with the corresponding pJK vectors for cellulase expression. Per lane, 5 μg total soluble protein (TSP) (A) or 15 μg TSP (B) were loaded. (A) Depicts a blot of the cell lysate with the corresponding Coomassie stained gel. (B) Depicts a blot of the cell pellet fraction with the corresponding Coomassie stained gel.
Mentions: Samples were harvested from E. coli cultures and N. tabacum leaves and the resulting extracts were used directly for western blotting and activity assays. To detect possible negative effects on E. coli growth behavior related to cellulase expression, OD600 at the end of the cultivation time was measured and compared to the empty vector control. Table 2 shows the average OD600 measured from four independent cultivations. This showed no clear negative effect of the cellulase expression on the final cell density. The bacterial extracts yielded bands in the western blot indicating the successful expression of all genes except those encoding exocellulases E3 and E6 (Figure 2A). The mobility of each band was consistent with the theoretical mass of the corresponding protein (Table 3), showing the successful expression of full-length proteins. Furthermore, each construct produced a single band on the blot suggesting the absence of protein degradation products. The blot for the cell pellet fraction (Figure 2B) showed bands only for BglC, E1, E4, and E5 indicating that a part of the protein remains insoluble. An additional band was observed for E1 indicating a slight degradation or a potential isoform of the enzyme when present in the insoluble fraction. For the exocellulases E3 and E6 as well as the endocellulase E2 there were no bands observed.

Bottom Line: Only the β-glucosidase showed high activity against 4-MUC.In contrast, all the plant-derived enzymes were active against the respective model substrates.Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biology VII (Molecular Biotechnology), RWTH Aachen University Aachen, Germany.

ABSTRACT
The economic conversion of lignocellulosic biomass to biofuels requires in addition to pretreatment techniques access to large quantities of inexpensive cellulases to be competitive with established first generation processes. A solution to this problem could be achieved by plant based expression of these enzymes. We expressed the complete set of six cellulases and an additional β-glucosidase expressed from Thermobifida fusca in the bacterium Escherichia coli and in tobacco plants (Nicotiana tabacum). This was done to determine whether functional enzyme expression was feasible in these organisms. In extracts of recombinant E. coli cells, five of the proteins were detected by western blot analysis, but exocellulases E3 and E6 were undetectable. In the plant-based expression system we were able to detect all six cellulases but not the β-glucosidase even though activity was detectable. When E. coli was used as the expression system, endocellulase E2 was active, while endocellulases E1 and E5 showed only residual activity. The remaining cellulases appeared completely inactive against the model substrates azo-carboxymethyl-cellulose (Azo-CMC) and 4-methylumbelliferyl-cellobioside (4-MUC). Only the β-glucosidase showed high activity against 4-MUC. In contrast, all the plant-derived enzymes were active against the respective model substrates. Our data indicate that some enzymes of bacterial origin are more active and more efficiently expressed in plants than in a bacterial host.

No MeSH data available.


Related in: MedlinePlus