Recipient Organization
DELAWARE STATE UNIVERSITY
1200 NORTH DUPONT HIGHWAY
DOVER,DE 19901
Performing Department
Agriculture & Natural Resources
Non Technical Summary
There is global effort lead by the United States and private industries to develop alternative sources of energy with the long-term goal of sustaining energy from agricultural products. Considerable progress has been made in ethanol production from agricultural products derived from corn, sugarcane, and cassava. Cassava (Manihot esculenta), also known as manioc, which is grown worldwide (particularly in Africa, South America and most of Southeast Asia) as a food source for billion of people, raising the possibility that it could be used globally to alleviate dependence on fossil fuels. Cassava tubers are an excellent source of carbohydrates with 20-40% starch content. Cassava starch costs 15-30% less to produce per acre than corn starch making cassava an attractive and strategic source of renewable energy. Cassava grows in diverse environments, especially extremely harsh climatic conditions, and its starch is already being used for large-scale ethanol production in many Asian and African countries. Conventionally, starch is liquefied using a-amylase and amylopullulanase with glucoamylase, before the sugars are used as feedstock for ethanol fermentation. The enzymes used in this process are expensive and are produced with genetically engineered microbes. Engineering cassava tubers to express hyperthermophilic starch-hydrolyzing enzymes and removing the need to add costly enzymes should help reduce the cost associated with starch breakdown into sugars and increase ethanol yields. We anticipate that the efficiency of cassava starch degradation will lead to substantial environmental benefits as well as 10¬15% cost savings in cassava-based ethanol production. The aims of this initial project are (1) to genetically engineer cassava plants to express Pyrococcus furiosus a-amylase and amylopullulanase in their tuberous roots, and (2) to determine whether the starch extracted from these transgenic tubers can autohydrolyze. Depending on the outcome of this feasibility phase, future work will include fine-tuning the expression of these two genes in cassava tubers and expressing the complete P. furiosus enzymatic starch degradation apparatus (Le., a-amylase, amylopullulanase, a-glucosidase, and glucoamylase) in cassava tubers, characterizing the best transgenic cassava lines for carbohydrate composition and starch self-processing properties, and testing the performance of transgenic cassava plants under field conditions in the United States, Puerto Rico, Afica, or South-East Asia. Once this approach is validated in cassava, it will be applied to other starch-rich crops to lower the corn demand for ethanol production. This project will also strengthen our plant biotechnology research and teaching programs currently significantly underdeveloped within our institution and will provide a unique opportunity for undergraduate and graduate students in biological and agricultural sciences and other related disciplines to acquire various skills in plant biotechnology and bioenergy research.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
Our goal is to produce enhanced cassava plants and design novel tuberous roots for better biofuel economy and to open up new markets for cassava. In a first feasibility phase, we will focus on expressing a-amylase and amylopullunase genes in cassava. The outcome of this initial phase will allow us to develop standard grant proposals for phase II. In phase II, and beyond, we plan to engineer the complete P. furiosus starch degradation enzymatic apparatus in cassava tubers, improve the expression of these genes in transgenic tubers, evaluate the ethanol conversion efficiency of starch derived from these transgenic tubers, initiate field trials of these plants in the US Virgin Islands, and start expanding this approach to other starch-rich crops of economical importance to the US such as corn and sweet sorghum, sweet potatoes, potatoes, wheat. Therefore, the specific objectives of this two-year proposal are: 1) to design and construct tubers-specific single-gene expression cassettes with the P. furiosus a-amylase and amylopullulanase genes; 2) to transform cassava plants with these starch-hydrolyzing gene expression cassettes and recover transgenic plants and tubers; 3) to evaluate transgenic cassava tubers for a-amylase and amylopullulanase expression and corresponding enzymatic activity; 4) to evaluate the liquefaction and autohydrolysis ability of starch in these transgenic cassava plants/tubers; and 5) to evaluate the effects of biophysical parameters such as temperature and pH on a-amylase and amylopullulanase activities during liquefaction of engineered cassava tubers. Designing and constructing tuber-specific single gene expression cassettes with amylase and amylopullulanase genes as well as transforming and recovering engineered cassava plants and tubers will be completed during the first and second year of the project. Towards the end of the second year, transgenic tubers will be collected from various lines to evaluate the expression of amylase and amylopullulanase and the corresponding enzyme activities. During the same period, the ability of starch derived from these transgenic lines to liquefy and autohydrolysis as well as the effects of biophysical parameters on the activity of these enzymes during these processes will be evaluated. Cassava could become a land-based feedstock for US ethanol production, especially in US territories and states where ecological and climatic conditions are favorable for cassava growth (e.g., Virgin Islands, Florida, Hawaii, Puerto-Rico, and Alabama County). We expect to develop genetically engineered cassava tubers that produce their own enzymes with the potential to break down starch into sugar. We also expect that the recombinant enzymes will break down starch only during the starch-processing stage, and not in the living tubers, hereby avoiding using expensive, commercial microbial starch-liquefying enzymes during ethanol production.
Project Methods
Starch hydrolyzing gene expression cassettes will be constructed by placing the pyrococcus furiosus amylase and amylopullulanase genes with or without their secretory signal sequence under patatin promoter to target their expression in cassava tubers. The two genes will be PCR-amplified from plasm ids PS4 and PSK211. These genes with or without their signal peptides will be cloned behind the patatin promoter in pUC19. The genes correctly fused to a promoter will be sub-cloned into the PCAMBIA 2300 binary vector to generate all the chimeric constructs to be used in this study. The expression cassettes will be confirmed in E. coli using restriction digestions and sequencing, and mobilized into disarmed Agrobacterium strain LBA4404 by electroporation and the resulting transformed Agrobacteria are used as vector systems for cassava transformation. The chimeric constructs will be introduced into friable embryogenic cassava calli produced from somatic embryos of cassava genotype TMS 60444 with A. tumefaciens. Regenerated putative transgenic plantlets will then be propagated in vitro and hardened. Tuberous roots will be harvested from 7-months old transgenic, green house-established plants for biochemical analysis. Alpha-amylase and amylopullulanase activities will be measured in crude enzyme extracts prepared from engineered tubers. The enzyme extracts from transgenic tubers will be used to hydrolyze starch from other crops as well as pullulan, glycogen, amylose, amylopectin, and oligosaccharides using published method. Substrate specificity of both enzymes will be determined by analyzing the carbohydrate profile of all hydrolysis products using High-Performance Anion Exchange Chromatography. Hydrolysis products will be identified and quantified using the PEAK II computer software. Alpha-amylase and amylopullulanase activities will also be determined using the 3-5-dinitrosalicyclic acid (DNS) method. The pH and temperature characteristics and heat stability of recombinant a-amylase and amylopullulanase in transgenic cassava tubers will be evaluated. Starch liquefaction in dried and milled transgenic tubers will be tested by incubating a tuber-derived flour suspension at 90C for 10 min, and then at 70C for 2h. The reducing sugar released will be quantified using the DNS method. Hydrolysis products will be analyzed by High-Performance Anion Exchange Chromatography. To determine the extent to which the starch in transgenic tubers can be autohydrolyzed at high temperature and converted into sugars, flour from the transgenic tubers will be prepared as a 1 % slurry that will be heated at 90C for various lengths of time, centrifuged, and both supernatant and precipitate used to determine starch and sugar content. If time permits, the liquefied milled cassava tubers will be saccharified with a commercial glucoamylase, and the saccharified samples will be tested for yeast ethanol fermentation. Data generated will be compared with previous works and publications. Results will be published in high quality and high impact journals. Papers will be presented at scientific meetings, symposia and seminars. US Patents will be filed for any breakthrough results