In planta differential targeting analysis of Thermotoga maritima Cel5A and CBM6-engineered Cel5A for autohydrolysis

Mahadevan, Shobana Arumugam; Wi, Seung Gon; Kim, Yeon Ok; Bae, Hyeun‐Jong

doi:10.1007/s11248-010-9464-8

Cited by 23 publications

(22 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The activity and accumulation of BglB and Cel5A with transit peptide in the chloroplasts of tobacco plants have been reported previously (Jung et al , 2010; Kim et al , 2010; Mahadevan et al , 2011), as has XylII accumulation via dual targeting to chloroplasts and peroxisomes in A. thaliana (Bae et al , 2006). The current study successfully coexpressed the cellulase genes linked by the 2A self-cleavage sequence [RsBglB-2A-RaCel5A] and [RsXylII-2A-RaCel5A] and observed synergistic activity of the proteins expressed in the [RsBglB-2A-RaCel5A] lines and Cel5A that was ~8% of TSP in transgenic [RsXylII-2A-RaCel5A] line 20.…”

Section: Discussionsupporting

confidence: 70%

Synergistic effects of 2A-mediated polyproteins on the production of lignocellulose degradation enzymes in tobacco plants

Lee

Jung

et al. 2012

Journal of Experimental Botany

Self Cite

View full text Add to dashboard Cite

Cost-effective bioethanol production requires a supply of various low-cost enzymes that can hydrolyse lignocellulosic materials consisting of multiple polymers. Because plant-based enzyme expression systems offer low-cost and large-scale production, this study simultaneously expressed β-glucosidase (BglB), xylanase (XylII), exoglucanase (E3), and endoglucanase (Cel5A) in tobacco plants, which were individually fused with chloroplast-targeting transit peptides and linked via the 2A self-cleaving oligopeptideex from foot-and-mouth disease virus (FMDV) as follows: [RsBglB-2A-RaCel5A], [RsXylII-2A-RaCel5A], and [RsE3-2A-RaCel5A]. The enzymes were targeted to chloroplasts in tobacco cells and their activities were confirmed. Similarly to the results of a transient assay using Arabidopsis thaliana protoplasts, when XylII was placed upstream of the 2A sequence, the [RsXylII-2A-RaCel5A] transgenic tobacco plant had a more positive influence on expression of the protein placed downstream. The [RsBglB-2A-RaCel5A] and [RsE3-2A-RaCel5A] transgenic lines displayed higher activities towards carboxylmethylcellulose (CMC) compared to those in the [RsXylII-2A-RaCel5A] transgenic line. This higher activity was attributable to the synergistic effects of the different cellulases used. The [RsBglB-2A-RaCel5A] lines exhibited greater efficiency (35–74% increase) of CMC hydrolysis when the exoglucanase CBHII was added. Among the various exoglucanases, E3 showed higher activity with the crude extract of the [RsBglB-2A-RaCel5A] transgenic line. Transgenic expression of 2A-mediated multiple enzymes induced synergistic effects and led to more efficient hydrolysis of lignocellulosic materials for bioethanol production.

show abstract

Section: Discussionsupporting

confidence: 70%

Synergistic effects of 2A-mediated polyproteins on the production of lignocellulose degradation enzymes in tobacco plants

Lee

Jung

et al. 2012

Journal of Experimental Botany

Self Cite

View full text Add to dashboard Cite

show abstract

“…The production time and expression efficiencies of spray‐based cellulase production surpass those achieved in plants stably transformed with nonreplicating expression vectors. For example, for endoglucanase Cel5A from T. maritima , expression levels of 4.5% and 5.2% were reported (Mahadevan et al ., ) and (Kim et al ., ), respectively) in stably transgenic tobacco plants compared to 12.5% of TSP in transiently transfected N. benthamiana . High accumulation of endoglucanase cel9A (about 40% of TSP) achieved by chloroplast transformation resulted in severe mutant phenotype of transgenic plants (Petersen and Bock, ).…”

Section: Discussionmentioning

confidence: 92%

A novel and fully scalable Agrobacterium spray‐based process for manufacturing cellulases and other cost‐sensitive proteins in plants

Hahn

Giritch

Bartels

et al. 2014

Plant Biotechnology Journal

View full text Add to dashboard Cite

SummaryTransient transfection of plants by vacuum infiltration of Agrobacterium vectors represents the state of the art in plant-based protein manufacturing; however, the complexity and cost of this approach restrict it to pharmaceutical proteins. We demonstrated that simple spraying of Nicotiana plants with Agrobacterium vectors in the presence of a surfactant can substitute for vacuum inoculation. When the T-DNA of Agrobacterium encodes viral replicons capable of cell-to-cell movement, up to 90% of the leaf cells can be transfected and express a recombinant protein at levels up to 50% of total soluble protein. This simple, fast and indefinitely scalable process was successfully applied to produce cellulases, one of the most volume-and cost-sensitive biotechnology products. We demonstrate here for the first time that representatives of all hydrolase classes necessary for cellulosic biomass decomposition can be expressed at high levels, stored as silage without significant loss of activity and then used directly as enzyme additives. This process enables production of cellulases, and other potential high-volume products such as noncaloric sweetener thaumatin and antiviral protein griffithsin, at commodity agricultural prices and could find broad applicability in the large-scale production of many other cost-sensitive proteins.

show abstract

“…Some studies have investigated biological means to reduce the cost of enzyme dosing by altering the cell wall composition or polysaccharide structure of a selected feedstock (Taylor et al, 2008; Mahadevan et al, 2011). For example, transgenic maize plants expressing cell wall degrading enzymes had 141 and 172% higher glucose and xylose yields, respectively, following enzymatic hydrolysis, compared with control plants.…”

Section: Engineering Plants To Enhance Cellulosic Biomassmentioning

confidence: 99%

Emerging Technologies for the Production of Renewable Liquid Transport Fuels from Biomass Sources Enriched in Plant Cell Walls

2016

View full text Add to dashboard Cite

Plant cell walls are composed predominantly of cellulose, a range of non-cellulosic polysaccharides and lignin. The walls account for a large proportion not only of crop residues such as wheat straw and sugarcane bagasse, but also of residues of the timber industry and specialist grasses and other plants being grown specifically for biofuel production. The polysaccharide components of plant cell walls have long been recognized as an extraordinarily large source of fermentable sugars that might be used for the production of bioethanol and other renewable liquid transport fuels. Estimates place annual plant cellulose production from captured light energy in the order of hundreds of billions of tons. Lignin is synthesized in the same order of magnitude and, as a very large polymer of phenylpropanoid residues, lignin is also an abundant, high energy macromolecule. However, one of the major functions of these cell wall constituents in plants is to provide the extreme tensile and compressive strengths that enable plants to resist the forces of gravity and a broad range of other mechanical forces. Over millions of years these wall constituents have evolved under natural selection to generate extremely tough and resilient biomaterials. The rapid degradation of these tough cell wall composites to fermentable sugars is therefore a difficult task and has significantly slowed the development of a viable lignocellulose-based biofuels industry. However, good progress has been made in overcoming this so-called recalcitrance of lignocellulosic feedstocks for the biofuels industry, through modifications to the lignocellulose itself, innovative pre-treatments of the biomass, improved enzymes and the development of superior yeasts and other microorganisms for the fermentation process. Nevertheless, it has been argued that bioethanol might not be the best or only biofuel that can be generated from lignocellulosic biomass sources and that hydrocarbons with intrinsically higher energy densities might be produced using emerging and continuous flow systems that are capable of converting a broad range of plant and other biomasses to bio-oils through so-called ‘agnostic’ technologies such as hydrothermal liquefaction. Continued attention to regulatory frameworks and ongoing government support will be required for the next phase of development of internationally viable biofuels industries.

show abstract

In planta differential targeting analysis of Thermotoga maritima Cel5A and CBM6-engineered Cel5A for autohydrolysis

Cited by 23 publications

References 35 publications

Synergistic effects of 2A-mediated polyproteins on the production of lignocellulose degradation enzymes in tobacco plants

Synergistic effects of 2A-mediated polyproteins on the production of lignocellulose degradation enzymes in tobacco plants

A novel and fully scalable Agrobacterium spray‐based process for manufacturing cellulases and other cost‐sensitive proteins in plants

Emerging Technologies for the Production of Renewable Liquid Transport Fuels from Biomass Sources Enriched in Plant Cell Walls

Contact Info

Product

Resources

About