Specific activity and utility of recombinant cellobiohydrolase II (Cel6A) produced in maize endosperm.

Cellobiohydrolase II (CBH II) is an exo-glucanase that is part of a fungal mixture of enzymes from a wood-rot fungus, Trichoderma reesei. It is therefore difficult to purify and to establish a specific activity assay. The gene for this enzyme, driven by the rice Os glutelin promoter, was transformed into High II tissue culture competent corn, and the enzyme accumulated in the endosperm of the seed.

Development of a highly sensitive glucose nanocomposite biosensor based on recombinant enzyme from corn.

Background: Enzymes are biocatalysts that play a vital role in the production of biomolecules. Plants can be a valuable and cost-effective source for producing well-structured recombinant enzymes. Glucose is one of the most important biological molecules, providing energy to most living systems.

Report on the SCRA Nuts and Bolts Workshop II: case studies of citrus greening, Ultra-low Gossypol Cotton, and blight tolerant, low-acrylamide potato.

To be commercialized and grown in the US, genetically engineered (GE) crops typically go through an extensive food, feed, and environmental safety assessment process which, in certain instances, requires complex consultations with three different US regulatory agencies. Many small market, niche, and specialty crops have been genetically engineered using the modern tools of recombinant DNA but few have been commercialized due to real or perceived regulatory constraints. This workshop discussed the practical aspects of developing dossiers on GE specialty, niche, or small-market crops/products for submission to US regulatory agencies.

Effects of Oligosaccharides Isolated From Pinewood Hot Water Pre-hydrolyzates on Recombinant Cellulases.

Loblolly pine residues have enormous potential to be the raw material for advanced biofuel production due to extensive sources and high cellulose content. Hot water (HW) pretreatment, while being a relatively economical and clean technology for the deconstruction of lignocellulosic biomass, could also inhibit the ensuing enzymatic hydrolysis process because of the production of inhibitors. In this study, we investigated the effect of oligosaccharide fractions purified from HW pre-hydrolyzate of pinewood using centrifugal partition chromatography (CPC) on three recombinant cellulolytic enzymes (E1, CBHI and CBHII), which were expressed in the transgenic corn grain system.

Plant-based biofuels.

This review is a short synopsis of some of the latest breakthroughs in the areas of lignocellulosic conversion to fuels and utilization of oils for biodiesel. Although four lignocellulosic ethanol factories have opened in the USA and hundreds of biodiesel installations are active worldwide, technological improvements are being discovered that will rapidly evolve the biofuels industry into a new paradigm. These discoveries involve the feedstocks as well as the technologies to process them.

Over-expression of the cucumber expansin gene (Cs-EXPA1) in transgenic maize seed for cellulose deconstruction.

Plant cell wall degradation into fermentable sugars by cellulases is one of the greatest barriers to biofuel production. Expansin protein loosens the plant cell wall by opening up the complex of cellulose microfibrils and polysaccharide matrix components thereby increasing its accessibility to cellulases. We over-expressed cucumber expansin in maize kernels to produce enough protein to assess its potential to serve as an industrial enzyme for applications particularly in biomass conversion.

Purification and characterization of recombinant Cel7A from maize seed.

The corn grain biofactory was used to produce Cel7A, an exo-cellulase (cellobiohydrolase I) from Hypocrea jecorina. The enzymatic activity on small molecule substrates was equivalent to its fungal counterpart. The corn grain-derived enzyme is glycosylated and 6 kDa smaller than the native fungal protein, likely due to more sugars added in the glycosylation of the fungal enzyme.

Assessment of field-grown cellulase-expressing corn.

Transgenic plants in the US and abroad generated using genetic engineering technology are regulated with respect to release into the environment and inclusion into diets of humans and animals. For crops incorporating pharmaceuticals or industrial enzymes regulations are even more stringent. Notifications are not allowed for movement and release, therefore a permit is required.

Development of a green binder system for paper products.

Background: It is important for industries to find green chemistries for manufacturing their products that have utility, are cost-effective and that protect the environment. The paper industry is no exception. Renewable resources derived from plant components could be an excellent substitute for the chemicals that are currently used as paper binders.

Risk assessment and regulation of molecular farming - a comparison between Europe and US.

In this article, the general principles of genetically modified (GM) plant risk assessment and the regulatory framework for contained use and open field production of plant-made pharmaceuticals/plant-made industrials (PMP/PMI) are described. While significant progress has been made for the containment grown (plant cell culture) production of PMPs, with the first regulatory approval made by the FDA in 2012, the commercialization of medicinal or industrial products produced in the field has yet to emerge in either Europe or the US. In the current paper, we discribe the regulatory environment in Europe and the US surrounding GM crops, and provide case studies for experimental field releases of PMP and PMI producing plants in both regions.

Transcriptome analysis of embryo maturation in maize.

Background: Maize is one of the most important crops in the world. With the exponentially increasing population and the need for ever increased food and feed production, an increased yield of maize grain (as well as rice, wheat and other grains) will be critical. Maize grain development is understood from the perspective of morphology, hormone responses, and storage reserve accumulation.

Heterologous expression of cellobiohydrolase II (Cel6A) in maize endosperm.

The technology of converting lignocellulose to biofuels has advanced swiftly over the past few years, and enzymes are a significant constituent of this technology. In this regard, cost effective production of cellulases has been the focus of research for many years. One approach to reach cost targets of these enzymes involves the use of plants as bio-factories.

Recombinant protein production in plants: challenges and solutions.

In a recent presentation at the 2010 International Association for Plant Biotechnology meeting, Dr. Richard Flavell (Ceres, Malibu, CA, USA) motivated the plant community to act quickly and with purpose to move a multitude of traits into crop plants to improve their productivity. Current progress toward understanding of plants is too slow and will not achieve our communal goal of doubling agricultural productivity by 2050.

Manipulating corn germplasm to increase recombinant protein accumulation.

Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal.

Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed.

Ethanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.

Manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed.

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed.

Expression of the sweet protein brazzein in maize for production of a new commercial sweetener.

The availability of foods low in sugar content yet high in flavour is critically important to millions of individuals conscious of carbohydrate intake for diabetic or dietetic purposes. Brazzein is a sweet protein occurring naturally in a tropical plant that is impractical to produce economically on a large scale, thus limiting its availability for food products. We report here the use of a maize expression system for the production of this naturally sweet protein.

Criteria for high-level expression of a fungal laccase gene in transgenic maize.

Expression of industrial enzymes in transgenic plants offers an alternative system to fungal fermentation for large-scale production. Very high levels of expression are required to make the enzymes cost-effective. We tested several parameters to determine the best method for achieving high levels of expression for a fungal laccase gene.

Analysis of the maize polyubiquitin-1 promoter heat shock elements and generation of promoter variants with modified expression characteristics.

The maize polyubiquitin-1 (Ubi-1) promoter is one of a few select promoters used to express foreign genes in monocots, such that recombinant proteins can be produced at commercially viable levels. Modifying the activity, specificity and responsiveness of such promoters provides a means to achieve desired levels and patterns of expression of genes encoding target products. Ubi-1 is constitutively expressed but is further induced by heat shock.

Where, oh where has my protein gone?

A recent publication by R. Chikwamba and colleagues highlights interesting issues in recombinant protein expression in transgenic plants. In the study they expressed a bacterial antigen in maize seed and obtained aberrant localization data.

Selecting the fruits of your labors.

Generation of transgenic plants exhibiting traits of interest requires the marriage of several technologies including gene transfer, selection and plant regeneration. Variety is the driver for these technologies because of the breadth of plant species requiring modification. A new selectable marker gene, pflp, has been applied to the recovery of orchid plants that exhibit resistance to a major bacterial disease that plagues the orchid industry.

Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants.

Bovine trypsin (EC 3.4.21.

Corn as a production system for human and animal vaccines.

The synthesis of selected antigens in plants and their oral delivery has great potential for reducing the costs of vaccine production and administration. The application of this technology requires antigen concentrations in final plant material to be uniform to ensure consistent dosing. In addition, antigen levels should be such as to allow the volume of each dose, containing a set amount of antigen, to be practical for oral delivery.

Monoclonal antibody manufacturing in transgenic plants--myths and realities.

The number and types of antibodies expressed in plants has increased steadily since the first reports of this accomplishment in the 1980s, illustrating the versatility of plants as a production system for antibodies. Many recent reviews have detailed the antibody forms that have been derived from plant expression systems. This contribution focuses on the remaining challenges to develop plant-derived therapeutic antibodies into products, and some of the progress that is being made in addressing these challenges.