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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 transgenic tobacco. Saccharification (72 hours at 50°C) of transgenic tobacco pretreated at 110°C, 140°C and 170°C with 100 mg/g cellulose loading of the commercial cellulase Spezyme CP at 100 mg of Spezyme per gram of biomass. Error bars represent standard deviations of triplicate sample analyses.
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Figure 3: Saccharification of transgenic tobacco. Saccharification (72 hours at 50°C) of transgenic tobacco pretreated at 110°C, 140°C and 170°C with 100 mg/g cellulose loading of the commercial cellulase Spezyme CP at 100 mg of Spezyme per gram of biomass. Error bars represent standard deviations of triplicate sample analyses.

Mentions: E1-expressing tobacco plants were pretreated in 1% (wt/vol) sulfuric acid for 10 minutes at three different temperatures, 110°C, 140°C and 170°C, resulting in a range of severities. Figure 3 illustrates that at 110°C and 140°C pretreatment temperatures, E1cd-transformed tobacco was about 10% more enzymatically digestible compared to the wild type or to wild-type biomass with exogenous E1cd added. Interestingly, the mixed wild-type/E1 sample is right between the E1cd-containing sample and the wild-type sample, supporting the observation that in planta-expressed E1cd required less severe pretreatment than the wild-type plant to achieve comparable sugar release. We also observed that at the highest pretreatment severity, 170°C, all of the samples exhibited a similar level of digestion. Presumably, at severe pretreatment levels, the improved conversion of the E1-containing tobacco becomes irrelevant as the conversion of the materials approaches a theoretical maximum.


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 transgenic tobacco. Saccharification (72 hours at 50°C) of transgenic tobacco pretreated at 110°C, 140°C and 170°C with 100 mg/g cellulose loading of the commercial cellulase Spezyme CP at 100 mg of Spezyme per gram of biomass. 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 3: Saccharification of transgenic tobacco. Saccharification (72 hours at 50°C) of transgenic tobacco pretreated at 110°C, 140°C and 170°C with 100 mg/g cellulose loading of the commercial cellulase Spezyme CP at 100 mg of Spezyme per gram of biomass. Error bars represent standard deviations of triplicate sample analyses.
Mentions: E1-expressing tobacco plants were pretreated in 1% (wt/vol) sulfuric acid for 10 minutes at three different temperatures, 110°C, 140°C and 170°C, resulting in a range of severities. Figure 3 illustrates that at 110°C and 140°C pretreatment temperatures, E1cd-transformed tobacco was about 10% more enzymatically digestible compared to the wild type or to wild-type biomass with exogenous E1cd added. Interestingly, the mixed wild-type/E1 sample is right between the E1cd-containing sample and the wild-type sample, supporting the observation that in planta-expressed E1cd required less severe pretreatment than the wild-type plant to achieve comparable sugar release. We also observed that at the highest pretreatment severity, 170°C, all of the samples exhibited a similar level of digestion. Presumably, at severe pretreatment levels, the improved conversion of the E1-containing tobacco becomes irrelevant as the conversion of the materials approaches a theoretical maximum.

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