Abhaya Dandekar - US grants

9407177 McDonald This project is to advance and broaden our knowledge in the areas of plant cell biotechnology, ribosome inactivating proteins (RIPs), and plant cell bioprocess engineering. This project has three specific goals, which include: (1) characterization of the transformation of plant cell cultures by the plant pathogen Agrobacterium rhizogenes; (2) quantification of cell growth and RIP production in suspension culture; and (3) purification and characterization of proteins obtained from the cell cultures. RIPs are naturally occurring plant proteins thought to be ubiquitous in the plant kingdom, and are currently receiving considerable attention for their antiviral, antitumor and abortifacient properties. They are also being considered for their potential use in immunotoxin preparation and for their ability to confer resistance to plant pathogens. ***

The objective of this project is to produce a plant virus based expression system combined with plant cell culture as a means for producing human therapeutic proteins. In particular, this project will genetically engineer host plant cells so that recombinant plant virus amplicons (self-replicating viral RNA containing a foreign gene) can be produced intracellularly, under the control of a chemically or metabolically-inducible promoter. The major goals of this project include: (1) the design of the recombinant viral gene constructs which contain all of the necessary components for high level production of viral mRNA, efficient viral replication, efficient foreign gene expression and extracellular targeting, 2) the design and creation of transgenic plant cell lines utilizing tightly regulated, chemically and/or metabolically inducible promoters which restrict viral production under non-induced conditions and allow high level transcription of the recombinant viral RNA under induction conditions, and 3) optimization of plant cell bioreactor conditions and operating strategies during growth and induction to maximize volumetric productivity of the product and minimize recovery/purification costs. This project will focus on the production of two model proteins (green fluorescent protein (GFP) and human a1-antitrypsin (AAT)) in tobacco cell cultures utilizing a cucumber mosaic virus (CMV) amplicon under the control of specific chemical and metabolically regulated promoters in a membrane bioreactor.

This project involves a multidisciplinary approach combining molecular biology and virology strategies with quantitative process engineering. In addition, an integrated training program consisting of a collaborative, multidisciplinary team approach to research, combined with coursework, a focused study group, seminars, retreats and an industrial internship to provide breadth in biotechnology, is part of this project. Graduate students, postdoctoral researchers and undergraduates will participate in this training program.

A multi-institutional Integrative Graduate Traineeship (IGERT) program, entitled Collaborative Research and Education in Agricultural Technologies and Engineering (CREATE) links the University of California at Davis with Tuskegee University in a training program with the overarching theme of the utilization of plants for the production of industrial non-food products and biopharmaceuticals. This program brings together a diverse group of faculty and graduate students from plant sciences, molecular biology and engineering to work together in interdisciplinary teams that will tackle critical societal challenges with applications in the following areas: 1) rapid vaccine production and cost-effective therapeutics, 2) biofuels and biorefineries, and 3) phytoremediation. The IGERT training program is aimed at developing specific skills that advance complex, interdisciplinary technologies. Novel components of the CREATE program include (1) the establishment of a Masters-to-PhD bridging program, (2) the development and implementation of a new graduate lecture course and two summer laboratory short courses on "Plant Transformation Methods" and "Recovery and Purification of Plant-Derived Products," and (3) industrial research internship opportunities in the United States as well as international research internship opportunities in Ireland at the University of Ireland, Maynooth and Teagasc Oak Park Research Centre. The broader impact of the CREATE program is the creation of a diverse group of research and educational leaders who will have knowledge of the fundamental principles and applications of plant science, biotechnology and bioprocess engineering as well as an understanding of the broader issues (environmental impact, public/societal views and global impact) of the field. The CREATE program emphasizes integrated training in plant sciences, molecular biology and engineering, to train the research, educational, business, regulatory, and policy leaders of the future who will help solve society's most pressing problems related to health, energy sustainability, and environmental stewardship. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

This NSF award by the Biotechnology, Biochemical and Biomass Engineering program supports the development of technologies and processes for efficient, rapid and scalable production of cellulase enzymes in plant tissues. To meet the requirements of the Energy Independence and Security Act of 2007, conversion of cellulosic biomass into advanced biofuels will require a significant amount of cellulose degrading enzymes at a low cost. Current methods for production of cellulose and hemicellulose degrading enzymes rely primarily on submerged fungal fermentation and are expensive, energy intensive, generate carbon dioxide, and consume cellulosic feedstock for growth of the production microorganisms. In this project, multiple thermostable cell wall degrading enzymes will be produced in Nicotiana benthamiana (a relative of tobacco) plants and harvested leaves, utilizing novel transient expression systems. Advantages of the transient approach over in-planta production of enzymes in stably transformed plants are that it is rapid, can utilize wild type (non-transgenic) plant biomass, and that the plant growth and enzyme production stages are decoupled and can be separately optimized.
Intellectual merit: This research project will improve fundamental bioprocess engineering understanding of transient agroinfiltration processes, plant viral amplicon expression systems, recovery and characterization of plant-made enzymes, scale-up considerations in transient agroinfiltration processes, and techno-economic and environmental assessment of alternative bioprocessing strategies for production of cellulase enzymes.
Broader impact: The benefits of the proposed research to society are 1) cost-effective, energyefficient and environmentally-sound technologies that will not only be useful for the production of cellulase and hemicellulase enzymes to meet the U.S biofuels mandates but also lead to "greener" and more efficient bioproduction technologies for the industrial enzyme industry, 2)enhanced fundamental process understanding of recombinant protein production using transient agroinfiltration in plants which will accelerate growth of companies utilizing similar technologies for vaccine and therapeutics production, 3) broadening participation of women and underrepresented minorities through proactive recruitment and interdisciplinary research and educational training opportunities at the interface of plant science, biotechnology and bioprocess engineering, and 4) broad curricular impact by incorporating research findings into existing and new lecture and laboratory courses.

This NSF RAPID project will contribute to the development of new biomanufacturing processes for proteins used in the treatment of Ebola that will dramatically increase the production capacity during the current crisis. Currently, antibodies against Ebola (the ZMapp antibodies) are produced in a relative of the tobacco plant. Antibody production in the whole plant is relatively slow. In the proposed work, investigators will move production from the whole plant into plant cells that can then be grown in large fermenters, much like other biopharmaceutical products. By optimizing the manufacturing conditions, the investigators believe that they can obtain a 100 fold increase in productivity for the same mass of plant material. The work will contribute to the Nation's ability to respond to other global health crises in the future by creating new biomanufacturing platforms and the capacity to rapidly increase production.

Technical: This NSF Rapid Response Research (RAPID) project will support the development of a new biomanufacturing process for the mixture of three monoclonal antibodies (ZMapp) that has been used to successfully treat patients infected with the Ebola virus. Currently, ZMapp is produced in Nicotiana benthamiana (N. benthamiana) in whole plants using vacuum agroinfiltration. This mode of production is dependent upon transient agroinfiltration of indoor or greenhouse grown plants, and is severely limited in production capacity relative to traditional stainless steel fermenter types of biomanufacturing processes. However, production of the ZMapp antibodies in more traditional hosts such as Chinese hamster ovary cells (CHO) had led to less effective monoclonal antibodies (lower survival rates and higher doses needed in animal studies) compared to the N. benthamiana produced antibodies. The researchers in this project will develop methods for large-scale suspension culture production of ZMapp antibodies from N. benthamiana cells. These methods will employ novel techniques for culturing agroinfiltrated plant cells in batch and semi-continuous modes. The work performed will enable the rapid production of the antibodies in the same types of large-scale facilities available for other biotherapeutics, while retaining the desirable antibody properties associated with proteins produced from N. benthamiana. The project is appropriate for the RAPID mechanism based on the urgency of need to develop scalable biomanufacturing methods to produce Ebola therapeutics that are already known to be effective. The project will contribute to our understanding and ability to design new biomanufacturing platforms that use plant cells, and enable the advance of synthetic biology in plant cells. Beyond the Ebola crisis, this work should help develop capacity for rapid biomanufacturing in response to future threats to global health.