Advanced enzyme production method
Technology
This technology and business are unique in that it enables mass production of enzymes in a safe geneticlally modified host plant in commercially significant quantities with a small agricultural footprint. 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.
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.
Concerns over finite petroleum reserve require development of alternative energy resources. Lower emission of green house gases from alternative energy resources is also highly desirable. The fact that corn ethanol produces more green house gas emissions than gasoline and that cellulosic ethanol from nonfood crops produces less green house gas emissions than electricity or hydrogen, highly favours production of ethanol from cellulosic biomass. However, biofuel production from lignocellulosic materials is a challenging problem because of the multifaceted nature of raw materials and lack of technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. The high costs of enzyme production and the tremendous amount of enzymes needed to hydrolyse pretreated biomass are often considered as key obstacles in the commercial lignocellulosic ethanol industry (Margeot et al., 2009).
The technology has been proven on a laboratory scale, and needs further work for commercialization and expansion into additional lignocellulosic feedstock substrates. It is our plan to raise funds to perform this work and to grow this critical technology.
Commercialization of genetically modified organisms is a regulated industry. USDA APHIS and BRS permits and processes must be followed to ensure all products are safely contained within the confines of open cultivation. Coordinating experienced growers with USDA genetically modified plants and growing techniques is a must.
US Patent # Title5,693,507 Genetic Engineering of Plant Chloroplasts6,680,426 Genetic Engineering of Plant Chloroplasts7,129,391 Universal Chloroplast Integration And Expression Vectors, Transformed Plants And Products Thereof (‘Universal Vector Application’)7,135,620 Genetic Engineering of Plant Chloroplasts11/938,331 Multiple Gene Expression for Engineering Novel Pathways and Hyperexpression of Foreign Proteins in Plants (DIV of US Pat. # 7,294,506)10/741,379 Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection12/396,382 Production and Use of Plant Degrading Materials
Financials
Funds raised will be adequet to break even in 29 months. This includes heavy front loading to accommodate research fees, engineering costs and fees, permits, licenses, USDA APHIS and BHS approved greenhouse, expenses, payroll and materials used in the commercial plant.
This buisness plan is to scale up to commercial and forward onto industrial scale means for growing genetically modified host plants to be used as lving factories for enzyme and specialized protein production. These proteins and enzymes may be used to increase efficiences and decrease overall costs associated with alternative fuel production, and for the production of other organic products.
Future milestones for this project include:
Establishment of primary research facility. This phase will require $3.7M over the first year
First revenues after initial commerical product are available.
Commercialize enzyme manufacture process for existing enzyme cocktails. This phase will require $1.1M over the following 18 months
Develop new enzyme cocktails for new feedstock markets. This phase will require an additional $1.1M.
We are planning on three rounds of project funding.
The first round would establish the research facility, and develop key knowledge, processes and technologies for transgenic enzyme development
The second round would expand production capabiility to ensure first revenue and continue research into next generation feedstock.
Lastly, the third round would allow the expansion of manufacturing capability and sales into the global market.
The archetype customer is the biofuel refinery who specializes in cellulosic ethanol, cellulosic biobutanol and other commerical or industrial chemical processing companies.
Particularly strong growth in biofuels consumption is projected for the United States, where production of biofuels increases from 0.3 million barrels per day in 2006 to 1.9 million barrels per day in 2030, supported by legislation in the Energy Independence and Security Act of 2007 that mandates increased U.S. use of biofuels. Other regions with sizable projected increases in biofuels production include OECD Europe, non-OECD Asia, and Central and South America. Those regions, together with the United States, account for 75 percent of the world increase in biofuels production." The US EPA Renewable Fuel Standard adds "The Renewable Fuel Standard program will increase the volume of renewable fuel required to be blended into gasoline from 9 billion gallons in 2008 to 36 billion gallons by 2022.
We have aquired LOIs from a number of US and Brazil customers interested in exclusive and non-exclusive supply arrangements. Customer names are available under NDA.
Industrial ethanol is a mature market. However, due to the rapid growth in fuel ethanol, the market dynamics in the industrial and fuel ethanol markets are actively changing. The passage of the 2007 US Energy Bill has been a substantial driver of market dynamics. Key suppliers in this market include domestic and imported fermentation producers, as well as domestic and imported synthetic producers. The Distributor focus covers the Industrial Ethanol Market and the Fuel Ethanol Market.
Sales and marketing efforts will follow BBRI recommendations and include direct marketing efforts, as well as through strategic parnerships with ethanol equipment manufacturers, providing raw materials required to meet the RFS2 market mandates and improve plant efficiencies.
There are a number of competitors in the commercial enzyme business, but only three are strategic competitors in the renewable fuels sector. These include Genencor, Danisco, Gevo, Ayris, and the market leader Novozymes. Our ability to manufacture product that is both technologically superior with lower manufacturing costs will allow GRES to enter the market quickly.
This project harnesses recent advances in genetic engineering for a novel approach to deliver low-cost biofuels to a rapidly expanding commodities market. This second-generation biofuels technology is a major advance over traditional methods of production because it can convert previously unusable, valueless biomass into marketable energy products and co-products. This technology does not require dedicated “energy crops.” Thus, it is considerably more environmentally friendly and socially acceptable than first-generation technologies currently serving the biofuels market. Significant revenues from co-products are also possible using this proprietary technology.
Our business model includes building upon the existing technologies and developing a commercial production capability to generate and sustain revenues and profit, while progressivly increasing the product portfolio. By positioning GRES as an innovative biotechnology company, it can generate additional revenues leading to eventual exit.
The products developed and innovated by GRES will include an expanding product line of tunable enzyme cocktails for the hydrolysis of cellulosic feedstock substrates into various renewable fuels including ethanol, butanol and other industrial chemicals.
