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In planta expression of A. cellulolyticus Cel5A endocellulase reduces cell wall recalcitrance in tobacco and maize.

Brunecky R, Selig MJ, Vinzant TB, Himmel ME, Lee D, Blaylock MJ, Decker SR - Biotechnol Biofuels (2011)

Bottom Line: Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion.Tobacco and maize plants were healthy and developed normally compared with the wild type (WT).Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biosciences Center, National Renewable Energy Laboratory, 1617 Cole Boulevard, MS 3323, Golden, CO 80401, USA. roman.brunecky@nrel.gov.

ABSTRACT
The glycoside hydrolase family 5 endocellulase, E1 (Cel5A), from Acidothermus cellulolyticus was transformed into both Nicotiana tabacum and Zea mays with expression targeted to the cell wall under a constitutive promoter. Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion. Tobacco and maize plants were healthy and developed normally compared with the wild type (WT). After thermochemical pretreatment and enzyme digestion, transformed plants were clearly more digestible than WT, requiring lower pretreatment severity to achieve comparable conversion levels. Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall.

No MeSH data available.


Related in: MedlinePlus

E1 expression in planta. Western blot analysis of wild-type (WT) and E1-transformed tobacco and corn stover.
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Figure 1: E1 expression in planta. Western blot analysis of wild-type (WT) and E1-transformed tobacco and corn stover.

Mentions: Both antibody- and activity-based assays were used to estimate the content of E1cd in stover and tobacco (data not shown). Initial Western blot analyses performed by extracting ~4 mg of milled stover with 50 μL of either 100% ethylene glycol at 80°C for 2 hours or 100% ethylene glycol followed by buffer (20 mM sodium acetate, 100 mM NaCl, pH 5.0) showed no bands (data not shown). Increasing the extraction severity by direct boiling of biomass in NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, Carlsbad, CA, USA) gave a strong band for the E1-1 stover sample which was the correct molecular weight for E1cd (Figure 1). In addition, for stover, a serial twofold dilution series Dot blot estimated the E1cd content of E1-1 to be approximately 3 ng/mg biomass (data not shown), which was a reasonable approximation for the Western blot analysis as well. The E1-7 sample was not detected by either antibody method; however, the 4-methylumbelliferyl-β-D-cellobiose (MUC) activity assay estimated the E1-7 level to be about 10-fold less than that of E1-1 (Figure 2). The MUC assay revealed E1cd levels of approximately 0.3 ng/mg biomass for E1-1 and 0.03 ng/mg biomass for E1-7. Wild-type stover showed no presence of E1 by either method. For tobacco, E1cd levels were approximately 1,000-fold higher than those in the transgenic stover. Western blot analysis estimated E1cd levels in tobacco to be approximately 3.1 μg/mg biomass. The higher level of E1 in tobacco is likely due to the use of the cauliflower mosaic virus (CaMV) promoter to drive expression in both maize and tobacco, as CaMV has been shown to be more active in dicotyledon plants.


In planta expression of A. cellulolyticus Cel5A endocellulase reduces cell wall recalcitrance in tobacco and maize.

Brunecky R, Selig MJ, Vinzant TB, Himmel ME, Lee D, Blaylock MJ, Decker SR - Biotechnol Biofuels (2011)

E1 expression in planta. Western blot analysis of wild-type (WT) and E1-transformed tobacco and corn stover.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037329&req=5

Figure 1: E1 expression in planta. Western blot analysis of wild-type (WT) and E1-transformed tobacco and corn stover.
Mentions: Both antibody- and activity-based assays were used to estimate the content of E1cd in stover and tobacco (data not shown). Initial Western blot analyses performed by extracting ~4 mg of milled stover with 50 μL of either 100% ethylene glycol at 80°C for 2 hours or 100% ethylene glycol followed by buffer (20 mM sodium acetate, 100 mM NaCl, pH 5.0) showed no bands (data not shown). Increasing the extraction severity by direct boiling of biomass in NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, Carlsbad, CA, USA) gave a strong band for the E1-1 stover sample which was the correct molecular weight for E1cd (Figure 1). In addition, for stover, a serial twofold dilution series Dot blot estimated the E1cd content of E1-1 to be approximately 3 ng/mg biomass (data not shown), which was a reasonable approximation for the Western blot analysis as well. The E1-7 sample was not detected by either antibody method; however, the 4-methylumbelliferyl-β-D-cellobiose (MUC) activity assay estimated the E1-7 level to be about 10-fold less than that of E1-1 (Figure 2). The MUC assay revealed E1cd levels of approximately 0.3 ng/mg biomass for E1-1 and 0.03 ng/mg biomass for E1-7. Wild-type stover showed no presence of E1 by either method. For tobacco, E1cd levels were approximately 1,000-fold higher than those in the transgenic stover. Western blot analysis estimated E1cd levels in tobacco to be approximately 3.1 μg/mg biomass. The higher level of E1 in tobacco is likely due to the use of the cauliflower mosaic virus (CaMV) promoter to drive expression in both maize and tobacco, as CaMV has been shown to be more active in dicotyledon plants.

Bottom Line: Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion.Tobacco and maize plants were healthy and developed normally compared with the wild type (WT).Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biosciences Center, National Renewable Energy Laboratory, 1617 Cole Boulevard, MS 3323, Golden, CO 80401, USA. roman.brunecky@nrel.gov.

ABSTRACT
The glycoside hydrolase family 5 endocellulase, E1 (Cel5A), from Acidothermus cellulolyticus was transformed into both Nicotiana tabacum and Zea mays with expression targeted to the cell wall under a constitutive promoter. Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion. Tobacco and maize plants were healthy and developed normally compared with the wild type (WT). After thermochemical pretreatment and enzyme digestion, transformed plants were clearly more digestible than WT, requiring lower pretreatment severity to achieve comparable conversion levels. Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall.

No MeSH data available.


Related in: MedlinePlus