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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

Saccharification of unpretreated corn stover. Saccharification (24 hours at 50°C) of unpretreated corn stover with two loadings of Spezyme CP (15 and 100 mg/g cellulose) or T. reesei Cel7a (15 mg/g cellulose). Error bars represent standard deviations of triplicate sample analyses.
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Figure 6: Saccharification of unpretreated corn stover. Saccharification (24 hours at 50°C) of unpretreated corn stover with two loadings of Spezyme CP (15 and 100 mg/g cellulose) or T. reesei Cel7a (15 mg/g cellulose). Error bars represent standard deviations of triplicate sample analyses.

Mentions: When digestions utilizing commercial cellulase loadings of 15 mg/g to those loaded at 100 mg/g (Figures 4 and 5) were compared, the E1cd-stover sample with the high E1 expression level (E1-1) appeared to show, at 15 mg/g loading, a comparable or higher rate of conversion compared to the wild-type stover treated with the 100 mg/g loading under the same pretreatment conditions. We also assessed the enzymatic conversion of the corn stover without pretreatment or preincubation (Figure 6) and again observed that the E1-1 corn stover appeared to be 5% to 8% more digestible than the wild-type control. Utilizing an exoglucanase (Cel7A) instead of a commercial cellulase yielded comparable conversion levels for both the high and low E1 samples, both of which appeared to have a higher glucan conversion compared to the control and the control plus exogenous E1 (Figure 7).


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)

Saccharification of unpretreated corn stover. Saccharification (24 hours at 50°C) of unpretreated corn stover with two loadings of Spezyme CP (15 and 100 mg/g cellulose) or T. reesei Cel7a (15 mg/g cellulose). Error bars represent standard deviations of triplicate sample analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Saccharification of unpretreated corn stover. Saccharification (24 hours at 50°C) of unpretreated corn stover with two loadings of Spezyme CP (15 and 100 mg/g cellulose) or T. reesei Cel7a (15 mg/g cellulose). Error bars represent standard deviations of triplicate sample analyses.
Mentions: When digestions utilizing commercial cellulase loadings of 15 mg/g to those loaded at 100 mg/g (Figures 4 and 5) were compared, the E1cd-stover sample with the high E1 expression level (E1-1) appeared to show, at 15 mg/g loading, a comparable or higher rate of conversion compared to the wild-type stover treated with the 100 mg/g loading under the same pretreatment conditions. We also assessed the enzymatic conversion of the corn stover without pretreatment or preincubation (Figure 6) and again observed that the E1-1 corn stover appeared to be 5% to 8% more digestible than the wild-type control. Utilizing an exoglucanase (Cel7A) instead of a commercial cellulase yielded comparable conversion levels for both the high and low E1 samples, both of which appeared to have a higher glucan conversion compared to the control and the control plus exogenous E1 (Figure 7).

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