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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

Expression cassettes used for cellulase expression. Expression cassettes based on pJK contained sequences derived from the Nicotiana tabacum chloroplast genes for tRNA-Gly (trnG) and tRNA-Met (trnM) used as homologous regions for recombination into the chloroplast genome of N. tabacum after particle bombardment as well as copies of the plastid ribosomal promoter (prrn) for expression of selectable marker gene aminoglycoside 3′-adenylyltransferase (aadA) and the gene of interest (goi) using 3′rbcL (ribulose bisphosphate carboxylase large subunit from Chlamydomonas reinhardtii) UTR as 3′ untranslated region. The cassette for the transient expression experiments contained scaffold attachment regions (SARs) and the p35SS promoter controlling transgene expression with an additional retention signal for localization in the endoplasmic reticulum (ER) and pA35S as polyadenylation signal.
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Figure 1: Expression cassettes used for cellulase expression. Expression cassettes based on pJK contained sequences derived from the Nicotiana tabacum chloroplast genes for tRNA-Gly (trnG) and tRNA-Met (trnM) used as homologous regions for recombination into the chloroplast genome of N. tabacum after particle bombardment as well as copies of the plastid ribosomal promoter (prrn) for expression of selectable marker gene aminoglycoside 3′-adenylyltransferase (aadA) and the gene of interest (goi) using 3′rbcL (ribulose bisphosphate carboxylase large subunit from Chlamydomonas reinhardtii) UTR as 3′ untranslated region. The cassette for the transient expression experiments contained scaffold attachment regions (SARs) and the p35SS promoter controlling transgene expression with an additional retention signal for localization in the endoplasmic reticulum (ER) and pA35S as polyadenylation signal.

Mentions: Seven target genes were successfully amplified by PCR and transferred to the target vectors pJK and pTRAkc-ERH for bacterial and plant transformation, respectively (Figure 1). The vectors included a polyhistidine coding sequence that was added to the 3′-end of each coding region to generate a C-terminal His6 tag. Sequencing the new plasmids revealed some differences compared to previously published sequences (Lao et al., 1991; Jung et al., 1993; Zhang et al., 1995; Irwin et al., 2000; Lykidis et al., 2007). The endocellulase E2 gene featured a silent mutation (30V) whereas the endocellulase E1 gene included a nucleotide exchange causing an amino acid substitution (174R→G) and also a deletion that removed residue 944G. The genes for E3 and E6 also contained mutations that caused amino acid substitutions (437N→D for E3 and 194I→T for E6). The remaining two cellulase genes and the sequence for the β-glucosidase (BglC) were identical to published data. Because the detected mutations were present in multiple clones for each gene, we assumed they represented naturally occurring alleles rather than PCR artifacts.


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)

Expression cassettes used for cellulase expression. Expression cassettes based on pJK contained sequences derived from the Nicotiana tabacum chloroplast genes for tRNA-Gly (trnG) and tRNA-Met (trnM) used as homologous regions for recombination into the chloroplast genome of N. tabacum after particle bombardment as well as copies of the plastid ribosomal promoter (prrn) for expression of selectable marker gene aminoglycoside 3′-adenylyltransferase (aadA) and the gene of interest (goi) using 3′rbcL (ribulose bisphosphate carboxylase large subunit from Chlamydomonas reinhardtii) UTR as 3′ untranslated region. The cassette for the transient expression experiments contained scaffold attachment regions (SARs) and the p35SS promoter controlling transgene expression with an additional retention signal for localization in the endoplasmic reticulum (ER) and pA35S as polyadenylation signal.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Expression cassettes used for cellulase expression. Expression cassettes based on pJK contained sequences derived from the Nicotiana tabacum chloroplast genes for tRNA-Gly (trnG) and tRNA-Met (trnM) used as homologous regions for recombination into the chloroplast genome of N. tabacum after particle bombardment as well as copies of the plastid ribosomal promoter (prrn) for expression of selectable marker gene aminoglycoside 3′-adenylyltransferase (aadA) and the gene of interest (goi) using 3′rbcL (ribulose bisphosphate carboxylase large subunit from Chlamydomonas reinhardtii) UTR as 3′ untranslated region. The cassette for the transient expression experiments contained scaffold attachment regions (SARs) and the p35SS promoter controlling transgene expression with an additional retention signal for localization in the endoplasmic reticulum (ER) and pA35S as polyadenylation signal.
Mentions: Seven target genes were successfully amplified by PCR and transferred to the target vectors pJK and pTRAkc-ERH for bacterial and plant transformation, respectively (Figure 1). The vectors included a polyhistidine coding sequence that was added to the 3′-end of each coding region to generate a C-terminal His6 tag. Sequencing the new plasmids revealed some differences compared to previously published sequences (Lao et al., 1991; Jung et al., 1993; Zhang et al., 1995; Irwin et al., 2000; Lykidis et al., 2007). The endocellulase E2 gene featured a silent mutation (30V) whereas the endocellulase E1 gene included a nucleotide exchange causing an amino acid substitution (174R→G) and also a deletion that removed residue 944G. The genes for E3 and E6 also contained mutations that caused amino acid substitutions (437N→D for E3 and 194I→T for E6). The remaining two cellulase genes and the sequence for the β-glucosidase (BglC) were identical to published data. Because the detected mutations were present in multiple clones for each gene, we assumed they represented naturally occurring alleles rather than PCR artifacts.

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