Deborah Vicuna Requesens

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. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.

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. The application of this technology to plant biomass conversion for biofuels and biobased products has the potential for significantly lowering the cost of these products due to lower enzyme production costs. Cel6A, one of the two cellobiohydrolases (CBH II) produced by Hypocrea jecorina, is an exoglucanase that cleaves primarily cellobiose units from the non-reducing end of cellulose microfibrils. In this work we describe the expression of Cel6A in maize endosperm as part of the process to lower the cost of this dominant enzyme for the bioconversion process. The enzyme is active on microcrystalline cellulose as exponential microbial growth was observed in the mixture of cellulose, cellulases, yeast and Cel6A, Cel7A (endoglucanase), and Cel5A (cellobiohydrolase I) expressed in maize seeds. We quantify the amount accumulated and the activity of the enzyme. Cel6A expressed in maize endosperm was purified to homogeneity and verified using peptide mass finger printing.

Development of a green binder system for paper products.

BackgroundIt 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. Air laid pressed paper products that are typically used in wet wipes must be bound together so they can resist mechanical tearing during storage and use. The binders must be strong but cost-effective. Although chemical binders are approved by the Environmental Protection Agency, the public is demanding products with lower carbon footprints and that are derived from renewable sources.ResultsIn this project, carbohydrates, proteins and phenolic compounds were applied to air laid, pressed paper products in order to identify potential renewable green binders that are as strong as the current commercial binders, while being organic and renewable. Each potential green binder was applied to several filter paper strips and tested for strength in the direction perpendicular to the cellulose fibril orientation. Out of the twenty binders surveyed, soy protein, gelatin, zein protein, pectin and Salix lignin provided comparable strength results to a currently employed chemical binder.ConclusionsThese organic and renewable binders can be purchased in large quantities at low cost, require minimal reaction time and do not form viscous solutions that would clog sprayers, characteristics that make them attractive to the non-woven paper industry. As with any new process, a large-scale trial must be conducted along with an economic analysis of the procedure. However, because multiple examples of “green” binders were found that showed strong cross-linking activity, a candidate for commercial application will likely be found.

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. Major breakthroughs are necessary! Thus, high-throughput methods that couple gene identification and phenotype observations are required to put potential products into the hands of plant breeders to make varieties with good agronomic characteristics that will be approved by the regulatory agencies. These first improved crops must be on the market in the next 10 years, according to Flavell, in order to begin to meet our doubled productivity goals in 30 years. Because it takes approximately 10 years to produce a characterized variety from an identified gene and move it through product development and regulatory approval, we must begin now. Presumably, by employing the techniques in the following -chapters, we can do that.

Assessment of field-grown cellulase-expressing corn

AbstractTransgenic 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. However, growing under permit is cumbersome and more expensive than open, non- regulated growth. Thus, when the genetically engineered pharmaceutical or industrial crop is ready for scale-up, achieving non-regulated status is critical. Regulatory compliance in the US comprises petitioning the appropriate agencies for permission for environmental release and feeding trials. For release without yearly permits, a petition for allowing non-regulated status can be filed with the United States Department of Agriculture with consultations that include the Food and Drug Administration and possibly the Environmental Protection Agency, the latter if the plant includes an incorporated pesticide. The data package should ensure that the plants are substantially equivalent in every parameter except for the engineered trait. We undertook a preliminary study on transgenic maize field-grown hybrids that express one of two cellulase genes, an exo-cellulase or an endo-cellulase. We performed field observations of whole plants and numerous in vitro analyses of grain. Although some minor differences were observed when comparing genetically engineered hybrid plants to control wild type hybrids, no significant differences were seen.

Regulatory issues of biotechnologically improved plants

Publisher Summary Plant biotechnology can be defined in many ways, but it is most often the genetic engineering of plants through the use of recombinant DNA. This chapter focuses on regulatory requirements for genetically engineered (GE) plants. For scientists, particularly public sector and small company scientists, this is the most daunting challenge partially because a simple checklist of tasks does not exist, and even when the developer begins to see the light at the end of the tunnel, the regulators can request even more data and testing. Generally, regulations cover the environmental release of plants produced through genetic engineering. The goals of a regulatory system should be and often are flexibility, science-based rigor, stakeholder-responsiveness, nationally and internationally coordinated, and risk proportional. The chapter discuses primarily the U.S regulatory system, because the first steps toward regulating GE crops were taken in the United States in the 1970s. The regulatory system has evolved into a system in which regulations are approached on a case-by-case basis. However, a new paradigm is needed for data collection and coordination to simplify and standardize the petition process for federal agencies—fulfilling data needs for crop assessment while ensuring safety of the product. In the end, the ultimate goal of genetic engineering of plants is to benefit society through improvements in agricultural practices, plant health, and plant productivity.

Commercial Plant-Produced Recombinant Cellulases for Biomass Conversion

Cellulases must be one of the least expensive enzymes produced to be effective for biomass conversion, and they must be produced in extremely large quantities. Thus, the seed production system is the system of choice for a number of reasons including high protein accumulation in the target tissue, long-term stable storage, and little capital equipment required for production other than farming equipment. An endocellulase and an exocellulase have been produced and sold from corn grain demonstrating functionality of the system. High protein accumulation, stable storage, and high utility in industrial cellulose degradation have been demonstrated.

Production of Industrial Proteins in Plants

The plant production system is advantageous for industrial enzymes. Enzymes with large scale products that demand low cost manufacturing are the markets of choice for plants. The plant production system is also advantageous for products that are harmful to single cell systems, for example oxidation/reduction (redox) enzymes. Four classes of enzymes are discussed in this chapter—xylanases, redox enzymes, amylases and cellulases. Examples of each of these classes of enzyme have been produced in plants—some as demonstration projects, others with the intent to sell the product. The authors have chosen specific examples to describe the advantages of the plant system, issues that have arisen, and potential for addressing markets. These case studies illustrate the value of using plants for production with simple agricultural inputs of sunlight, nutrients and water. With the developing demand for biofuels and biobased products, large volume enzyme markets for processing agricultural materials are rapidly becoming a demand. The logical system for producing those enzymes is in co-products of the feedstock materials. Our examples below illustrate the system.