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Extramural Research

Final Report: Development of Biotechnology to Sustain the Production of Environmentally Friendly Transportation Fuel Ethanol from Cellulosic Biomass

EPA Grant Number: R826118
Title: Development of Biotechnology to Sustain the Production of Environmentally Friendly Transportation Fuel Ethanol from Cellulosic Biomass
Investigators: Ho, Nancy W. Y.
Institution: Purdue University
EPA Project Officer: Richards, April
Project Period: November 1, 1997 through October 31, 2000
Project Amount: $359,877
RFA: Technology for a Sustainable Environment (1997)
Research Category: Pollution Prevention/Sustainable Development



In the past 3 years, we accomplished a great deal for our Saccharomyces yeast-for-cellulosic ethanol fuel project under the support of this program. The most important accomplishment is that we have solved a serious problem that allowed us to develop effective stable genetically engineered cellulosic sugars-fermenting yeasts that can be distributed freely to industry now to produce ethanol from cellulosic biomass. In addition, this breakthrough allowed us to prove that our gene integration system for integrating multiple copies of multiple genes into the yeast chromosome is extremely effective and reliable. With this method, we expect that we can further improve our yeast to be more efficient in ethanol production as well as to be able to produce a variety of coproducts. Such further improved yeasts will make the production of cellulosic ethanol more cost-effective, and make the use of domestically produced ethanol to replace gasoline a step closer to reality.

We originally proposed three tasks to: (1) genetically engineer new Saccharomyces yeasts for effective cofermentation of glucose and xylose; (2) further improve our yeast for fermenting sugars to ethanol as well as for the production of high value coproducts; and (3) reduce the formation of byproducts during fermentation of sugars to ethanol.

Summary/Accomplishments (Outputs/Outcomes):

In the past 3 years, in addition to completing our original proposed research plan, we also carried out additional research. Our accomplishments are outlined below.

Major Accomplishments

  1. Successful improvement of the xylose-fermenting ability of the Saccharomyces yeasts, in addition to the 1400 yeast, transformed with pLNH32 plasmid.

  2. Successful development of another genetically engineered glucose-xylose-cofermenting Saccharomyces yeast, in addition to 1400(LNH-ST), containing stably integrated xylose-metabolizing genes capable of effective cofermentation of glucose and xylose.

  3. Successful demonstration of the stepwise integration mechanism of our new method for integration of multiple copies of multiple exogenous genes into the yeast chromosomes.

  4. Successful development of other genetically engineered Saccharomyces yeasts, strain 424A(LNH-ST), containing stably integrated xylose-metabolizing genes capable of effective cofermentation of glucose and xylose. In particular, strain 424A(LNH-ST) is as effective as 1400(LNH-ST).

  5. Successful demonstration that our stable genetically engineered yeasts can repeatedly coferment glucose and xylose (using pure sugars or sugars from cellulosic biomass hydrolysates) to ethanol with high efficiencies for numerous cycles and requiring very little nutrients.

  6. Completion of the cloning and overexpression of TAKLl, TKLl, and ADHl in glucose-xylose-cofermenting Saccharomyces yeasts and their effect on ethanol fermentation.

  7. Cloning and overexpression of genes encoding glucose transporters HXT 1, HXT 4, HXT 5, and HXT 7 in genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts.

  8. Cloning and overexpression of genes encoding yeast galactose transporter and E. coli xylose transporter, which are highly possible to be able to improve xylose transport in our genetically engineered yeast.

  9. Development of a new system for analyzing xylose transport in the Saccharomyces yeast.

  10. Analysis of the activity of the key enzymes present in yeast 424A(LNH-ST) during glucose and xylose fermentation as a guide for further improvement of our genetically engineered yeast for cellulosic ethanol production.

Additional Important Accomplishments

  1. 1. Genetic engineering of the Saccharomyces yeasts for the reduction of byproduct formation, particularly the formation of glycerol, during fermentation of sugars.

  2. 2. Cloning the yeast L-lactate oxidoreductase gene to make yeast able to use lactic acid as a carbon source.

  3. 3. Preliminary study of genetic engineering of yeast for much improved production of lactic acid from traditional substrates and potentially from cellulosic biomass. These preliminary results, coupled with our established reputation as experts for genetic engineering of microorganisms, resulted in attracting a company to provide funding (since 1999) to develop an effective yeast for lactic acid production. Lactic acid is the feedstock for the production of numerous environmentally friendly products, including biodegradable plastics. This project has been very successful, and we are now preparing a patent application.

Recent studies have proven that ethanol as a transportation fuel produces less air pollutants than gasoline. This environmentally friendly liquid fuel can be used directly as a neat fuel (100 percent) or as a blend with gasoline at various concentrations. The raw material used for the production of ethanol fuel is renewable and abundantly available domestically. Thus, the use of ethanol to supplement or replace gasoline not only reduces air pollution and ensures a cleaner environment, but also reduces the dependency of our Nation on imported foreign oil, protects our Nation's energy security, and reduces our Nation's trade deficit due to imported oil for the production of gasoline.

Cellulosic biomass is renewable, available at low cost, and existent in great abundance all over the world, especially in the United States. Cellulosic biomass is therefore an attractive feedstock for the production of ethanol fuel and numerous other industrial products by fermentation. However, extracts of various types of cellulosic biomass contain several extra sugar molecules such as xylose, mannose, galactose, and arabinose in various amounts. In particular, nearly all types of cellulosic biomass contain a substantial amounts of xylose. Various economic studies have indicated that effective conversion of xylose to ethanol is absolutely necessary for cellulosic biomass to be considered as an economical feedstock for biofuel ethanol production.

The ordinary Saccharomyces yeasts have been used for centuries for the production of ethanol from glucose and fructose and are the only microorganisms used for large-scale ethanol production by industry. Furthermore, Saccharomyces yeasts also are able to ferment all other hexoses sugars such as mannose and galactose, besides glucose and fructose, to ethanol. However, Saccharomyces yeasts are unable to ferment xylose. Since 1980, scientists worldwide (at least 10 known research groups from the United States, Europe, Japan; at least 6 such groups from this country) have been actively trying to develop genetically engineered Saccharomyces yeasts to ferment xylose. In 1993, we succeeded in the development of the world's first genetically engineered Saccharomyces yeasts, 1400(pLNH33) and 1400(pLNH32), which can effectively coferment glucose and xylose to ethanol. This was accomplished by cloning three xylose metabolizing genes, xylose reductase gene (XR), xylitol dehydrogenase gene (XD), and xylulokinase gene (XK), on a 2 m-based high-copy-number plasmid, pUCKml0, and transforming the Saccharomyces yeast 1400 with the resulting plasmids, designated pLNH 31, -32, -33, and -34 (collectively referred to as pLNH plasmids).

The development of the recombinant yeast 1400(pLNH-32) and 1400(pLNH33) described above was a great technological breakthrough, proving that yeast could be genetically engineered to effectively ferment xylose and to effectively coferment glucose and xylose simultaneously to ethanol. However, because these first groups of genetically engineered yeasts rely on genes cloned on a high-copy-number plasmid to provide them the ability to ferment xylose, these cloned genes are not stable under the conditions (nonselective condition) used by industry for the production of ethanol. Thus, we need to develop stable yeast with the cloned genes integrated into the yeast chromosome. We need to integrate all three genes, XR, XD, and XK, into the yeast chromosome, and we also need to integrate, according to our estimation, at least 10 copies of each of the three genes into the yeast chromosome to make the resulting yeast as effective as 1400(pLNH32) or 1400(pLNH-33) to coferment glucose and xylose to ethanol.

To overcome the second obstacle, in 1995, we succeeded in creating a super-stable genetically engineered glucose-xylose-cofermenting Saccharomyces yeast 1400(LNH-ST) by integrating multiple copies of the three-gene cassette, XD-XR-XK, into the yeast chromosome. The resulting stable recombinant yeast 1400(LNH-ST) is even slightly more effective in cofermenting glucose and xylose than 1400(pLNH32) or 1400(pLNH33). To develop the ideal stable recombinant yeast, we had to develop a new and much more effective method for integrating high-copy-number genes into the yeast chromosome.

According to our design, it should be relatively easy to convert most Saccharomyces yeasts, if not all of them, to effective glucose-xylose cofermenters with either one of our pLNH plasmids (e.g., pLNH3l, pLNH34) or to integrate multiple copies of XR-XD-XK cassette into the yeast chromosome by our method. This would allow the screening of yeasts that are more effective in converting glucose and xylose to ethanol. Particularly, this also would allow us to easily convert the best industrial glucose fermenting yeasts to effective glucose and xylose cofermenters.

Yeast is still easier to be converted to glucose-xylose cofermenting yeast by transforming with pLNH32 (or our other pLNH plasmids), rather than integrating the three-gene cassette XD-XR-XK into the yeast chromosome. Thus, for evaluating the effectiveness of any yeast for cofermenting glucose and xylose to ethanol, our approach is first to transform the yeast with pLNH32 and characterize the transformants for cofermentation, tolerance to ethanol and inhibitors, etc., before final conversion to stable recombinant yeast. However, to our surprise, more than 10 different Saccharomyces yeasts that we tested subsequently transformed with pLNH 32 or other pLNH plasmids and had poor activities for fermenting xylose. The best is only half as effective as 1400(pLNH32) in cofermenting glucose/xylose. We knew that it was not our original hypothesis that was incorrect, but something else interfering with the expression of the xylose-fermenting activity of most of these genetically engineered yeasts.

In 1998, we focused our study on solving this problem, and we succeeded. As we suspected, it was not because those yeasts could not effectively ferment xylose, but because most of these yeasts transformed with pLNH32 had higher tolerance to geneticin antibiotic and/or perhaps also contained higher copies of endogenous 2µ plasmids than the 1400 yeast. As a result, few copies of the pLNH plasmids were maintained in those latter yeasts under the same culturing conditions (in the presence of 50 µg of geneticin per mL of the medium). When we increased the concentration of the antibiotic, the xylose-fermenting capability increased considerably.

Due to this important finding, we found that, as we predicted, most yeast transformed with pLNH32 or other pLNH plasmids could coferment glucose and xylose with similar efficiencies as 1400(pLNH32). After solving this puzzle, we have succeeded in developing more super-stable genetically engineered Saccharomyces yeasts such as 259A(LNH-ST) and 424A(LNH-ST), with similar efficiencies in cofermenting glucose and xylose to ethanol as our first super-stable engineered Saccharomyces yeast, 1400(LNH-ST). This proves our theory that most wild type Saccharomyces yeasts can be genetically engineered for effective cofermentation of glucose and xylose with our technology. This also proves that our integration method is reliable and easy to carry out.

We also were able to demonstrate that our method for integrating multiple copies of genes in the host chromosome is truly unique. It is the only method that can integrate exogenous genes into the host chromosome in a controlled manner, and only as many copies required for maximal expression of the exogenous gene(s) are integrated. Too many extra copies of the integrated exogenous gene might pose an extra burden to the host cells. Furthermore, because it is known that even genes integrated into the ribosomal genes might not be totally stable, our method also compensates for the possible loss of the integrated gene and provides a mechanism to replenish the lost gene to maintain maximal expression of the integrated gene indefinitely.

There was a legal reason that we needed to make new stable yeasts for cellulosic ethanol production. In repaying the financial support that a company provided to us for the development of these yeasts, we agreed that the company would have the exclusive right to use and sublicense our first stable yeast 1400(LNH-ST) strain to others. Nevertheless, we retained the rights to develop more similar stable yeasts. These and other reasons described above provided enough
incentives for us to want to make more stable yeasts. In addition, the company that has the exclusive rights to our 1400(LNH-ST) yeast had been fighting a legal battle with another company over the rights to use our yeasts since 1997. Thus, for the future of cellulosic ethanol production, we had to make more such stable yeasts, which should at least have similar efficiency as 1400(LNH-ST) in fermenting cellulosic sugars to ethanol. Now we are proud to report that our new stable yeasts, 259A(LNH-ST) and 424A(LNH-ST), are nearly as effective as 1400(LNH-ST) in cofermenting glucose and xylose to ethanol, and we also are free from any legal restraints for licensing. We can license them to any company to produce ethanol from cellulosic biomass. If we have the time and resources, I am sure that we could develop more stable yeasts that might be more efficient than 1400(LNH-ST) in cofermenting glucose and xylose to ethanol.

In recognition of the importance of our work, we have received two very prestigious international awards: The R&D 100 Award (selected by R&D Magazine as one of the 100 most technologically significant new products of the year) in 1998, and the Discover Award in 1999 from Discover Magazine for technological innovation. We were awarded the Discover Award in part due to the work supported by the TSE program of EPA. Our work has been widely reported by media all over the world. Furthermore, Purdue University also issued a special news release in August 1998, featuring our genetically engineered yeast on its Web site (http://news.uns.purdue.edu/UNS/htm14ever/9808.Ho.yeast.html), which will remain there until August 2002.

We also have found that our stable recombinant yeasts can repeatedly coferment glucose and xylose (using pure sugars or sugars from cellulosic biomass hydrolysates) to ethanol with high efficiencies, for numerous cycles, requiring very little nutrients. Furthermore, we also have developed a set of conditions under which our engineered yeasts can efficiently produce ethanol from acid hydrolysates without requiring expensive detoxification. These additional important new findings, plus the fact that our genetically engineered yeasts can be programmed to produce numerous coproducts by additional genetic manipulation, will ensure the development of a cost-effective and profitable Saccharomyces yeast-based process for the production of biofuel ethanol from cellulosic biomass.

We also completed the study of the cloning and overexpression of TAKLl, TKLl (encoding transaldolase and transketolase respectively), and ADHl (encoding alcohol dehydrogenase) genes in glucose-xylose-cofermenting Saccharomyces yeasts and their effect on ethanol fermentation. This study served two purposes. One was to find out whether cloning of these genes might lead to direct improvement of yeast xylose fermentation, because the enzymes encoded by these three genes play important roles in fermenting of xylose to ethanol. The second purpose, which might be more important, was to further shed light on how best to genetically engineer a metabolic process in any organism for improved production of the metabolic products or byproducts. Our early work with the xylulose kinase gene (XK), which catalyzes an irreversible reaction, proved that with cloning and overexpression of the XK, we could overcome all the imperfections present upstream to the xylulokinase reaction. This could be a general rule for genetic engineering to improve the products and byproducts produced by a metabolic reaction.

Our results indicated that overexpression of these three genes in our genetically engineered yeast had very little effect on the efficiency of our yeast in cofermenting both glucose and xylose. Thus, our results seem to support that only overexpression of genes encoding enzymes catalyzing irreversible reactions might be important to improve the efficiency of a microorganism to produce special metabolic products through genetic engineering. This is an important task in our project supported by the TSE program, and we would like to see it continue without interruption.

In the last 3 years, we also accomplished a great deal of other important work that has been outlined in our 1998 and 1999 annual reports to the program, and most of them will not be repeated here. However, due to the flexibility of the TSE program, we were able to start or continue other important projects that we expected to be funded by other agencies or companies, but the funding came in late or did not materialize. This has enabled us to salvage a few very important projects, including:

  • Genetic engineering of yeast for lactic acid production. We carried out preliminary studies under EPA's support during the contract negotiations with a company. Now, the project is very successful. Not only are we in the process of a patent application, but we are confident that our genetically engineered lactic acid-producing yeast will be used for the production of lactic acid from renewable biomass, including cellulosic biomass, to be used as raw material for the production of biodegradable plastics, another environmentally important project.

  • Improvement of yeast xylose transport. We knew that improving xylose transport is the next most serious obstacle we need to overcome to further substantially improve the efficiency of our yeast's xylose fermentation. We had proposed this to another agency before we planned to apply for funding from the TSE program. We fully expected to be funded; however, it did not materialize. The Principal Investigator (PI) had resubmitted the proposed research to other agencies, and the fate was the same. We could not understand why such an important project did not receive support. However, improvement of yeast xylose transport is too important to the yeast-for-cellulosic ethanol project, and we must proceed. Therefore, last year we added our new approach for overcoming this obstacle to our project supported by the TSE program.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 16 publications 2 publications in selected types All 1 journal articles

Type Citation Project Document Sources
Journal Article Ho NWY, Chen ZD, Sedlak M, Brainard AP. Successful design and development of genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose from cellulosic biomass to fuel ethanol.. Advances In Biochemical Engineering/Biotechnology. 1999;65:163-192. R826118 (Final)
not available
Supplemental Keywords:

green chemistry, genetics, organism, microorganism, transportation, alternative fuel, sustainable energy, innovative technology, ethanol fuel., RFA, Industry Sectors, Scientific Discipline, Sustainable Industry/Business, Genetics, Environmental Chemistry, Sustainable Environment, Technology for Sustainable Environment, Transportation and Warehousing - NAIC 48-49, Environmental Engineering, environmentally friendly transportation fuel, Saccharomyces yeasts, biotechnology, ethanol, genetic engineering, gene cloning, alternative fuel, alternative energy source, innovative technology, industrial enzymes, pollution prevention, green chemistry

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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