DEVELOPMENT OF A SCALABLE PRODUCTION SYSTEM FOR EXPANSIN PROTEINS FOR USE IN CONVERTING CELLULOSIC BIOMASS TO BIOFUEL.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 0213519
• Grant No.: 2008-33610-18920
• Proposal No.: 2008-00054
• Project Start Date: May 15, 2008
• Project End Date: Oct 30, 2009
• Grant Year: 2008
• Program Code: [8.8]- Biofuels and Biobased Products

Recipient Organization
EXPANSYN TECHNOLOGIES, INC.
200 INNOVATION BLVD., SUITE 258-B
STATE COLLEGE,PA 16803

Performing Department
(N/A)

Non Technical Summary
In the 2007 State of the Union address, President Bush called for 35 billion gallons of alternative fuel use in the US by 2017 which would replace 20% of the nation's petroleum consumption. It is apparent that this goal cannot be achieved with existing technology and existing feedstocks alone. New technology is needed to improve the conversion of non-grain (cellulosic) feedstocks. In the US, total fuel ethanol production in 2006 reached 4.86 billion gallons - a 24% increase over 2005. Domestic demand for fuel ethanol rose 33% over 2005 to 5.4 billion gallons (Renewable Fuels Association, March 2007.) There are 114 ethanol biorefineries in operation in the US with an aggregate capacity of 5.6 billion gallons. Nearly all fuel ethanol is produced by fermentation of corn glucose (from corn grain) in the US. Presently, there are no commercial cellulosic ethanol biorefineries in operation (Renewable Fuels Association, February 2007.) Commercialization of ETI's proprietary technology will address present and future needs for the growing biofuels market. In the short-term, ETI's biocatalyst will be developed as an accessory protein used in conjunction with the most advanced cellulase technologies. Successful implementation of the proposed Phase I and subsequent research and development effort will result in the use of an ETI protein as a separate, plant-derived additive to cellulase during the hydrolysis of recalcitrant cellulose to glucose. Longer term protein development will shift from use in biomass conversion to functionality in a dedicated, genetically modified biofuel crop. Since expansin proteins are ubiquitous in plant cell walls, it is possible that, through genetic engineering, expression of a select expansin protein will enhance the accessibility of plant cellulose to enzymatic attack. Developments in this arena could lead to proprietary plant crops which encode the ETI protein. The present Phase I application will provide important fundamental insight into the technical feasibility of this approach.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
511	2410	1000	100%

Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
2410 - Cross-commodity research--multiple crops;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

biofuel

biomass

cellulase synergism

enzymatic hydrolysis

expansin

viral expression

Goals / Objectives
The proposed research will develop a process for the production and use of a plant protein to improve the performance of enzymes used in the conversion of recalcitrant cellulose to fuel ethanol. In research conducted at Penn State University, a novel class of cell wall-loosening proteins, named expansins, was discovered. Expansin itself lacks cellulytic activity, but it was found to enhance the hydrolytic digestion of pure cellulose by cellulases. We believe that expansins could prove to be potent and economical synergists for the industrial deconstruction of cellulose into sugars and then by fermentation into liquid fuel for transportation purposes. To make its way onto the proving ground of industrial scale testing, two important milestones for commercial feasibility need to be met: (a) a scaleable system for expansin production and delivery must be established and (b) the effectiveness of expansin as a cellulase synergist needs to be tested with real-world samples of cellulosic biomass, such as corn stover, switchgrass, and forestry residues. These goals in fact are intertwined because goal (b) requires substantial amounts of expansin, necessitating goal (a). This proposal outlines a research program to accomplish these goals, which if successful will constitute a major step towards commercialization of this potential method for reducing the cost of cellulosic conversion to biofuel. The technical objectives include: 1) Use the virus-based plant expression system to produce g-scale quantities of expansin (four different types) and assess the activity and stability of the proteins. 2) Test expansin-cellulase synergy with corn stover, switchgrass and poplar wood, as representatives of the real-world cellulosic biomass. 3) Develop protocols for storage and if necessary purification.

Project Methods
The proposed research will be carried out by Expansyn Technologies, Inc. Dr. Nuwan Sella Kapu, will be the project principal investigator. The proposed work for phase I is as follows: A. Clone expansin genes into the appropriate magnifection viral expression systems. B. Infect host plants (Nicotiana benthamiana and Nicotiana excelsior) with Agrobacterium tumefaciens carrying the expansin-expression replicon. C. Assess the time course of protein expression with antibody. D. Assess expansin activity by wall extension assays and cellulase synergism. E. Produce g level amounts of expansin and test for cellulase-synergy with real world forms of biomass. F. Assess stability and storage requirements (wet, dry, refrigeration, freezing); G. If necessary, develop purification protocols.

Progress 05/15/08 to 10/30/09

Outputs
OUTPUTS: Despite being a high-volume feedstock for biofuel production and providing an attractive alternative for lowering national dependence on petroleum, cellulosic biomass is a difficult and slow material to process with cellulolytic enzymes. Biomass recalcitrance causes cellulosic ethanol production to be an expensive process, with significant cost contributions arising from both the large amounts of enzymes required for acceptable conversion rates as well as the harsh pretreatment procedures needed to improve digestibility. In research conducted at Penn State University, a novel class of cell wall-loosening proteins named expansins was discovered and found to enhance the hydrolysis of pure cellulose by cellulases. In this USDA Phase I SBIR project, we proposed to express several expansin proteins in Nicotiana using a virus-based expression system. Selected expansin and expansin-related genes were successfully amplified and cloned into vectors for magnifection. These constructs were delivered into several varieties of Nicotiana benthamiana and Nicotiana excelsior through A. tumefaciens. Tissue samples were collected at a range of time points following plant infection, and expansin protein expression was tracked using immunoblotting. Different formulations of tobacco-expressed expansins were evaluated for synergy with cellulase using dilute sulfuric acid pretreated corn stover as the substrate. PARTICIPANTS: Nuwan Sella Kapu - Principal Investigator; Marta Manning - Research Scientist; Yesenia Soto - Technician; Pennsylvania State University - partner organization; A portion of the research work was performed in the laboratory of Dr. Daniel Cosgrove at Penn State under a research subcontract. All workers received valuable training in techniques for expression of viral vectors in plants. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Some issues were encountered with achieving acceptable levels of expression of expansin proteins in tobacco. This led to the need for troubleshooting and delayed some aspects of the research. In addition, some assays on the expressed proteins could not be performed due to insufficient supply of protein for the studies.

Impacts
The completed Phase I research has increased our understanding of transient over-expression of expansin proteins in plants and the use of these proteins as synergists for cellulose hydrolysis. Expansin genes were successfully cloned into the appropriate magnifection vectors and the constructs delivered into Nicotiana plants. Protein expression levels varied depending on the identity of the protein used for plant transfection. Different formulations of plant tissue expressing these proteins showed significant cellulase synergistic activity during enzymatic hydrolysis of dilute acid pretreated corn stover. Stability of different formulations to refrigerated and frozen storage was also evaluated. Some tissue formulations retained activity for up to two months of frozen storage. Potential future impacts of the research findings include the ability to lower enzyme costs associated with cellulose hydrolysis reactions through the addition of plant-expressed expansin proteins.

Publications

• No publications reported this period

Progress 05/15/08 to 05/14/09

Outputs
OUTPUTS: Despite being a high-volume feedstock for fuel production and providing an attractive alternative for lowering national dependence on petroleum, cellulosic biomass is a difficult and slow material to process with cellulolytic enzymes. Biomass recalcitrance causes cellulosic ethanol production to be an expensive process, with significant cost contributions arising from both the large amounts of enzymes required for acceptable conversion rates as well as the harsh pretreatment procedures needed to improve digestibility. In research conducted at Penn State University, a novel class of cell wall-loosening proteins named expansins was discovered and found to enhance the hydrolysis of pure cellulose by cellulases. In this USDA Phase I SBIR project, we proposed to: express several expansin proteins in Nicotiana using a virus-based expression system, use Agrobacterium tumefaciens to mediate delivery of the expansin genes into Nicotiana plants, evaluate protein expression, storage, and stability properties, and assess the function of the plant-expressed proteins as synergists for the saccharification of industrially-relevant types of biomass. Several expansin and expansin-related protein genes (CsEXPA1, ZmEXPB1, ZM2, and ZM3) were successfully amplified and cloned into vectors for magnifection, and were transformed into A. tumefaciens. Several varieties of Nicotiana benthamiana and Nicotiana excelsior were then infected with constructs. Tissue samples were collected at a range of time points following plant infection, and expansin protein expression was tracked using immunoblotting. Since ZM3 and CsEXPA1 expression levels were relatively higher than those of the other two proteins tested, we decided to focus on these two proteins for our activity studies. Crude tissue extracts from Nicotiana plants expressing either ZM3 or CsEXPA1 were evaluated for synergy with cellulase using dilute sulfuric acid pretreated corn stover as the substrate and standard National Renewable Energy Laboratory protocols for enzymatic hydrolysis of biomass. In order to evaluate different formulations and storage strategies for expansin-expressing tobacco, a similar synergy analysis was conducted using minimally processed ground tobacco leaves as additives in dilute sulfuric acid pretreated corn stover hydrolysis. PARTICIPANTS: Nuwan Sella Kapu - Principal Investigator; Marta Manning - Research Scientist; Yesenia Soto - Technician; Pennsylvania State University - partner organization; A portion of the research work was performed in the laboratory of Dr. Daniel Cosgrove at Penn State under a research subcontract. All workers received valuable training in techniques for expression of viral vectors in plants. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Some issues were encountered with achieving acceptable levels of expression of expansin proteins in tobacco. This led to the need for troubleshooting and delayed some aspects of the research. In addition, some assays on the expressed proteins could not be performed due to insufficient supply of protein for the studies.

Impacts
The completed Phase I research has increased our understanding of transient over-expression of expansin proteins in plants and the use of these proteins as synergists for cellulose hydrolysis. Expansin protein genes were successfully cloned into the appropriate magnifection vectors, transformed into A. tumefaciens, and infiltrated into Nicotiana plants. Protein expression levels varied depending on the identity of the protein used for plant transfection. Levels of ZM1 protein were low, while CsEXPA1, ZM2 and ZM3 expression levels obtained using the virus-based plant expression system were more promising. Crude extracts from plant tissue expressing these proteins showed significant cellulase synergistic activity during enzymatic hydrolysis of dilute acid pretreated corn stover. Minimally processed ground tissue preparations from ZM3-overexpressing plants were likewise found to be effective synergists. Stability of both types of preparations to refrigerated and frozen storage was also evaluated. Ground tissue preparations as well as whole leaves retained activity for up to two months of frozen storage. Potential future impacts of the research findings include the ability to lower enzyme costs for cellulose hydrolysis reactions through the addition of plant-expressed expansin proteins in crude form. In particular, expression of expansins in stably-transformed dedicated energy crops such as switchgrass could present a promising strategy for reducing biomass recalcitrance and decreasing costs associated with biofuel production.

Publications

• No publications reported this period