Source: UNIVERSITY OF CENTRAL FLORIDA submitted to NRP
CHLOROPLAST-DERIVED ENZYME COCKTAILS FOR HYDROLYSIS OF LIGNOCELLULOSIC WASTE BIOMASS AND RELEASE OF FERMENTABLE SUGARS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0218579
Grant No.
2009-39200-19972
Cumulative Award Amt.
$219,960.00
Proposal No.
2009-04081
Multistate No.
(N/A)
Project Start Date
Sep 1, 2009
Project End Date
Feb 28, 2013
Grant Year
2009
Program Code
[CC-X]- Florida Biomass to Biofuels Conversion Prg., FL
Recipient Organization
UNIVERSITY OF CENTRAL FLORIDA
12722 RESEARCH PARKWAY
ORLANDO,FL 32826
Performing Department
Molecular Biology & Microbiology
Non Technical Summary
Currently, corn ethanol provides 3% of the US gasoline supply but consumes 30% of US corn crop, increasing the price of corn. Therefore, cellulosic ethanol from waste biomass would be an ideal source for ethanol production but would require development of new technology. We have recently produced several biomass degrading enzymes in tobacco chloroplasts. Based on three cuttings of tobacco in one year, 49, 64 and 10,751 million units of three important enzymes can be obtained each year. Based on enzyme activity observed in plant crude extracts, there is no need for purification. Therefore, excluding processing cost, enzymes could be produced 1000-3000 fold less expensive than current commercial recombinant enzymes. We have demonstrated for the first time that plant-derived enzyme cocktails enhanced hydrolysis of wood or citrus peel, releasing more fermentable sugars than commercial cocktails. Therefore, in this project, we propose to express in tobacco chloroplasts different classes of biomass degrading enzymes including endoglucanases, exoglucanases, lipases, pectate lyases, xylanases, acetyl xylan esterases, beta glucosidases, mannannase, lignin peroxidase, arabinofuranosidase, etc from bacterial and fungal genes. We will also express enzymes that increase their access for enhanced hydrolysis like cutinase and swollenin (expansin). Enzyme cocktails will be optimized to hydrolyze a variety of waste biomass including corn stover, rye straw, wood pulp, switchgrass, sugar cane bagasse or citrus peel to release fermentable sugars. Commercial production of recombinant enzymes in fermentation systems is limited by both higher cost and lower production capacity. Both these concerns will be addressed by chloroplast-derived enzyme cocktails.      
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5111999104050%
5112499104050%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
1999 - Tobacco, general/other; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
OBJECTIVES 1.Isolate genes encoding biomass degrading enzymes from bacterial and fungal genomes 2.Create chloroplast vectors and evaluate enzyme functions in E. coli 3.Create and characterize tobacco transplastomic lines in experimental and commercial cultivars expressing biomass degrading enzymes 4.Create enzyme cocktails for degradation of different types of biomass and compare with commercial cocktails
Project Methods
In this project, we propose to express in chloroplasts different classes of biomass degrading enzymes including endoglucanases, exoglucanases, lipases, pectate lyases, xylanases, acetyl xylan esterases, beta glucosidases, mannannase, lignin peroxidase, arabinofuranosidase, etc from bacterial and fungal genes. We will also express enzymes like cutinase and swollenin to increase access for enzymes for enhanced hydrolysis. A novel PCR based method will be used to clone ORFs without introns from Trichoderma reesei or other fungal genomic DNA. Enzyme cocktails will be used for biomass degradation to produce fermentable sugars and for direct comparison of properties of enzymes produced via fermentation or in planta, using identical genes and regulatory sequences. Enzyme cocktails will be made to utilize a variety of waste biomass including corn stover, rye straw, wood pulp, switchgrass, sugar cane bagasse and citrus peel.

Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: Results were disseminated by publication in the Plant Biotechnology Journal and several news articles appeared in the public press regarding this publication or featured articles appeared in other journals(www.biofuelsjournal.com). PARTICIPANTS: Principal Investigator - Henry Daniell Post-doctoral Fellows - Dheeraj Verma, Nameirakpam Dolendro Singh, Shuangxia Jin TARGET AUDIENCES: Biomass, biofuels PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Low cost chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars Dheeraj Verma, Anderson Kanagaraj, Shuangxia Jin, Nameirakpam Dolendro Singh, Pappachan E Kolattukudy and Henry Daniell* Department of Molecular Biology and Microbiology, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Biomolecular Science Building, 4000 Central Florida Blvd, Orlando, FL 32816-2364, USA (daniell@mail.ucf.edu) It is widely recognized that biofuel production from lignocellulosic materials is limited by the lack of technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, mixtures of enzymes containing endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in E. coli or tobacco chloroplasts. A novel PCR based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10,751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3,100-fold and pectate lyase is 1,057 or 1,480 fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher specific activity, temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Plant extracts were free of inhibitory factors that decreased enzyme activity in E. coli extracts. Expression of individual enzymes facilitated development of different cocktails for hydrolysis of filter paper, pine wood or citrus peel. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3,625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails.

Publications

  • Verma, D., Kanagaraj, A., Jin, S., Singh, N.D., Kolattukudi, P.E. and Daniell, H. (2010). Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars, Plant Biotechnology Journal 8: 332-350.