Source: NORTHERN REGIONAL RES CENTER submitted to
PATHWAY ENGINEERING OF FUNGI FOR IMPROVED BIOPROCESS APPLICATIONS
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
USDA INHOUSE
Reporting Frequency
Annual
Accession No.
0408515
Grant No.
(N/A)
Project No.
3620-41000-110-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
May 3, 2004
Project End Date
May 2, 2009
Grant Year
(N/A)
Project Director
SKORY C D
Recipient Organization
NORTHERN REGIONAL RES CENTER
(N/A)
PEORIA,IL 61604
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
51115101020100%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
1510 - Corn;

Field Of Science
1020 - Physiology;
Goals / Objectives
The overall objective of this project is the genetic modification of filamentous fungi for improved synthesis of value added products. The primary emphasis will focus on improving the efficiency of lactic acid production by Rhizopus oryzae through metabolic engineering. Additionally, the techniques developed in this research will be used to assess the potential of using this industrially robust organism for directed protein expression.
Project Methods
Utilize metabolic engineering technologies to develop fungal strains with improved production capability for lactic acid. The general approach of this work is to sequentially increase enzymatic activities responsible for the production of lactic acid, while reducing those involved in the production of unwanted byproducts. The effects of these changes will be examined through traditional methods (i.e., fermentations, enzymatic activities, and northern data), as well as some of the newer genomic and metabolic flux analyses that will be incorporated into our work. Additionally, we will continue to develop methods for improved genetic recombination and RNA interference in Rhizopus. We will also design and employ strategies for the production and secretion of recombinant proteins of industrial interest using the fungus Rhizopus. Initial efforts of this work will include development and study of promoter/terminator constructs in Rhizopus. We will determine the importance of copy number compared to transcription promoter strength. Lastly, much of the work is to be focused on the necessary requirements for efficient folding and secretion. BSL-1 (or 1P) and Risk Group RG1 recertified April 17, 2008.

Progress 05/03/04 to 05/02/09

Outputs
Progress Report Objectives (from AD-416) The overall objective of this project is the genetic modification of filamentous fungi for improved synthesis of value added products. The primary emphasis will focus on improving the efficiency of lactic acid production by Rhizopus oryzae through metabolic engineering. Additionally, the techniques developed in this research will be used to assess the potential of using this industrially robust organism for directed protein expression. Approach (from AD-416) Utilize metabolic engineering technologies to develop fungal strains with improved production capability for lactic acid. The general approach of this work is to sequentially increase enzymatic activities responsible for the production of lactic acid, while reducing those involved in the production of unwanted byproducts. The effects of these changes will be examined through traditional methods (i.e., fermentations, enzymatic activities, and northern data), as well as some of the newer genomic and metabolic flux analyses that will be incorporated into our work. Additionally, we will continue to develop methods for improved genetic recombination and RNA interference in Rhizopus. We will also design and employ strategies for the production and secretion of recombinant proteins of industrial interest using the fungus Rhizopus. Initial efforts of this work will include development and study of promoter/terminator constructs in Rhizopus. We will determine the importance of copy number compared to transcription promoter strength. Lastly, much of the work is to be focused on the necessary requirements for efficient folding and secretion. Significant Activities that Support Special Target Populations Studies with recombinant yeast strains that produce lactic acid suggest that transport of the lactate anion across the plasma membrane is a rate limiting step and is critical to prevent acidification of the cytoplasm. Expression of native yeast lactate transporters in Saccharomyces isolates can improve this limitation under certain conditions. In an effort to test the effectiveness of a novel fungal lactate transporter previously isolated in our laboratory, we developed our own Saccharomyces strains that simultaneously express the Rhizopus lactate dehydrogenase (LdhA) and lactate permease (LacA) proteins. Comparisons with control strains that are only expressing the Rhizopus LdhA suggest that the LacA does not improve fermentative productivity of lactic acid in the strains used in this study. Therefore, we have continued examining other putative Rhizopus lactic acid transporters to find superior methods of lactate transport across the cell membrane. Two additional monocarboxylate transporters cloned from Rhizopus have been shown to be functional in Saccharomyces isolates. We are continuing to examine these membrane proteins in both yeast and Rhizopus isolates. Additionally, we have made substantial progress with our ability to perform site directed integration and gene replacement in Rhizopus strains. Using these techniques, we deleted each of the two pyruvate decarboxylase (PDC) genes in Rhizopus in an attempt to minimize the production of the fermentative by-product ethanol and increase the final yield of lactic acid. Each PDC gene was successfully deleted, but we were unable to completely eliminate the presence of nuclei containing functional PDC, presumably due to the lethality of this deletion. Technology Transfer Number of New/Active MTAs(providing only): 9

Impacts
(N/A)

Publications

  • Ibrahim, A.S., Gebermariam, T., Fu, Y., Lin, L., Husseiny, M.I., French, S. W., Schwartz, J., Skory, C.D., Edwards, J.E., Spellberg, B.J. 2007. The iron chelator deferasirox protects mice from mucormycosis through iron starvation. Journal of Clinical Investigation. 117(9):2649-2657.
  • Skory, C.D., Mertens, J.A., Rich, J.O. 2009. Inhibition of Rhizopus Lactate Dehydrogenase by Fructose 1,6-bisphosphate. Enzyme and Microbial Technology. 44(4):242-247.
  • Li, X., Skory, C.D., Cotta, M.A., Puchart, V., Biely, P. 2008. Novel Family of Carbohydrate Esterases, Based on Identification of the Hypocrea jecorina Acetyl Esterase Gene. Applied and Environmental Microbiology. 74(24):7482-7489.
  • Ma, L.-J., Ibrahim, A.S., Skory, C.D., Grabherr, M.G., Burger, G., Butler, M., Elias, M., Idnurm, A., Lang, F., Sone, T., Abe, A., Calvo, S.E., Corrochano, L.M., Engels, R., Fu, J., Hansberg, W., Kim, J.-M., Kodira, C. D., Koehrsen, M.J., Liu, B., Miranda-Saavedra, D., O'Leary, S., Ortiz- Castellanos, L., Poulter, R., Rodriguez-Romero, J., Ruiz-Herrera, J., Shen, Y., Zeng, Q., Galagan, J., Birren, B.W., Cuomo, C.A., Wickes, B.L. 2009. Genomic Analysis of the Basal Lineage Fungus Rhizopus oryzae Reveals a Whole-Genome Duplication. PLoS Genetics. 5(7):e1000549.


Progress 10/01/06 to 09/30/07

Outputs
Progress Report Objectives (from AD-416) The overall objective of this project is the genetic modification of filamentous fungi for improved synthesis of value added products. The primary emphasis will focus on improving the efficiency of lactic acid production by Rhizopus oryzae through metabolic engineering. Additionally, the techniques developed in this research will be used to assess the potential of using this industrially robust organism for directed protein expression. Approach (from AD-416) Utilize metabolic engineering technologies to develop fungal strains with improved production capability for lactic acid. The general approach of this work is to sequentially increase enzymatic activities responsible for the production of lactic acid, while reducing those involved in the production of unwanted byproducts. The effects of these changes will be examined through traditional methods (i.e., fermentations, enzymatic activities, and northern data), as well as some of the newer genomic and metabolic flux analyses that will be incorporated into our work. Additionally, we will continue to develop methods for improved genetic recombination and RNA interference in Rhizopus. We will also design and employ strategies for the production and secretion of recombinant proteins of industrial interest using the fungus Rhizopus. Initial efforts of this work will include development and study of promoter/terminator constructs in Rhizopus. We will determine the importance of copy number compared to transcription promoter strength. Lastly, much of the work is to be focused on the necessary requirements for efficient folding and secretion. Accomplishments IMPROVED TECHNIQUES FOR GENETIC MODIFICATION OF RHIZOPUS. The fungus Rhizopus is frequently used to convert, or ferment sugars obtained from agricultural crops to lactic acid. This natural product has long been utilized by the food industry and is also used for the manufacture of environmentally friendly products which include the biodegradable plastic, poly-lactic acid (PLA), and the chlorine-free solvent, ethyl lactate. In order to allow the market potential of lactic acid to continue expanding at the current rapid pace, it is important that the production costs are minimized by the development of new and improved technologies. We recently developed new methods of introducing DNA into the fungus Rhizopus and have used these techniques to modify the enzymes involved in the production of lactic acid. The results of this study will allow further strategies to be developed for the industrial utilization of this valuable organism, thereby benefiting the agricultural grower and ultimately the consumer. This work was performed under NP 306, Quality and Utilization of Agricultural Products, New Processes, New Uses, and Value-Added Foods and Biobased Products. Technology Transfer Number of New CRADAS and MTAS: 2 Number of Active CRADAS and MTAS: 6 Number of Non-Peer Reviewed Presentations and Proceedings: 3

Impacts
(N/A)

Publications

  • Mertens, J.A., Skory, C.D. 2007. Isolation and characterization of a second glucoamylase gene without a starch binding domain from Rhizopus oryzae. Enzyme and Microbial Technology. 40:874-880.
  • Li, X., Skory, C.D., Ximenes, E.A., Jordan, D.B., Dien, B.S., Hughes, S.R., Cotta, M.A. 2007. Expression of an AT-rich xylanase gene from the anaerobic fungus Orpinomyces sp. strain PC-2 in and secretion of the heterologous enzyme by Hypocrea jecorina. Applied Microbiology and Biotechnology. 74:1264-1275.
  • Mertens, J.A., Skory, C.D. 2007. Isolation and characterization of two genes that encode active glucoamylase without a starch binding domain from Rhizopus oryzae. Current Microbiology. 54:462-466.
  • Skory, C.D., Ibrahim, A.S. 2007. Native and modified lactate dehydrogenase expression in a fumaric acid producing isolate Rhizopus oryzae 99-880. Current Genetics. 52:23-33.
  • Ibrahim, A.S., Skory, C.D. 2006. Genetic manipulation of zygomycetes. In: Kavanagh, K. editor. Medical Mycology, Cellular and Molecular Techniques. New York, NY: John Wiley & Sons. p. 305-325.


Progress 10/01/05 to 09/30/06

Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Fossil fuels are by far the most exploited natural resource used today. The whole infrastructure of modern society is built upon oil, which has a limited reserve. Petroleum serves as the foundation for not only most of the USA's energy needs, but also for many products, such as solvents, organic acids, and numerous polymers and plastics. In 2004, the USA consumed about 5.7 billion barrels of petroleum with approximately 2-5% being used for the production of plastics and solvents (Department of Energy (DOE) Petroleum Supply Annual 2004). Although many benefits to society are derived from the use of petroleum, potential problems, in the form of litter, landfill depletion, environmental pollution, global warming, and increased atmospheric carbon dioxide are associated with its use. Means to help alleviate these problems include the development of viable technologies for the production of industrial chemicals using renewable feedstocks (e.g., starch or cellulosic biomass) and the development of more efficient fermentation processes. Finding new uses for agricultural crops and improving the efficiency of how we utilize them is imperative for competing in today's global market. The broad objective of the CRIS Work Unit (CWU) is to develop improved technologies and novel systems for increasing yields and reducing costs of converting agricultural commodities/residues to value-added products (e.g., biofuels and organic acids) through enzyme and fermentation technology. The CWU concentrates primarily on studying the genetic control mechanisms for production of several metabolites and enzymes by the filamentous fungus Rhizopus oryzae. This fungus is used for large- scale manufacture of lactic acid. However, problems exist with fermentations because of synthesis of several unwanted byproducts that must be separated from the final product and decrease the overall yield of lactic acid. This fungus is also recognized as a robust organism that is routinely used for industrial enzyme production (e.g., glucoamylase, lipase, peptidase). Currently, its use is limited by the enzymes that the organism naturally produces. We have developed technologies that can now be used to genetically manipulate this fungus so that a host of useful proteins can be produced. This research contributes to the objectives of National Programs 306, Quality and Utilization of Agricultural Products. Corn and its processing byproducts (e.g., corn stover and corn fiber) will initially serve as the primary agricultural crop for this research, although technology is applicable to alternative crops. Customers of this work include: producers/consumers of lactic acid or its end-products (e.g., polylactic acid (PLA) plastic); industries requiring enzymes (e.g., biofuels, biomass conversion, food and feed, specialty chemicals); corn processors; new markets for agricultural commodities; agricultural community; scientific community with interests in fungal metabolic engineering; and general public. Cooperative Research and Development Agreements (CRADA) with appropriate companies are currently used to transfer products of this research. Much of the research focuses on the production of lactic acid because of the potential market growth of the biodegradable plastic, polylactic acid, and solvent ethyl lactate. Domestic production of lactic acid is greater than 50,000 tons/yr and will likely increase exponentially if the growth for PLA and ethyl lactate develops as expected. However, fermentation efficiency must be improved to ensure the economic feasibility of the anticipated market expansion and to ensure that the U. S. maintains its leadership role. Additionally, considerable effort is being directed towards new technologies for improved enzyme production, which currently are estimated to have annual sales in excess of $1 billion. The ability to genetically improve the production of these enzymes, as well as other novel recombinant enzymes (e.g., cellulase, xylanase), is expected to have significant implications. Furthermore, the genetic knowledge gained from this work can easily be adapted to aid research with numerous other fungi that have industrial value. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2004) Test feasibility of using Rhizopus oryzae for expression of desired proteins. Continue to modify expression of various genes involved in the synthesis of lactic acid and various unwanted byproducts. Year 2 (FY 2005) Begin study of protein secretion pathways for Rhizopus. Continue study of synthesis pathways for unwanted products formed during lactic acid production. Develop improved methods of integration and gene-knockout. Year 3 (FY 2006) Design improved methods of protein secretion. Apply technology of gene integration and gene-knockout to ameliorate production of unwanted by-products. Initiate development of biochemical and analytic tools for monitoring the metabolic state of Rhizopus during fermentation. Initiate work to investigate inhibitors of lactic acid production in Rhizopus. Year 4 (FY 2007) Utilize new analytical techniques to study modified strains and identify new targets for further improvements in lactic acid production and protein synthesis. Begin testing of pilot and large scale fermentations of modified strains. Identify targets to overcome inhibition of lactic acid production. Year 5 (FY 2008) Test improved protein production systems using several enzymes with industrial interest. Study methods to improve product stability during lactic acid production. Utilize technology of gene modification to improve lactic acid production in the presence of inhibitors. Continue pilot and large scale testing of modified strains. 4a List the single most significant research accomplishment during FY 2006. INHIBITION OF GENE EXPRESSION USING RNA INHIBITION - performed under NP 306, Quality and Utilization of Agricultural Products. Lactic acid is produced by fermentation technology using renewable agricultural products and has significant potential for market growth as a result of new interest in the biodegradable plastic, PLA. Rhizopus is a filamentous fungus that is currently used for industrial scale production of lactic acid, but also accumulates several unwanted by-products that decrease the potential yield and necessitate further purification. We have identified methods to decrease expression of genes and should be able to apply this technology to eliminate by-products forming through other metabolic pathways. The results of this work are currently being exploited for the development of recombinant strains that will be tested by a major lactic acid producer. This accomplishment directly addresses National Program 306 - Quality and Utilization of Agricultural Products (100%), in particular Component 2, New Processes, New Uses, and Value-Added Foods and Biobased Products. 4b List other significant research accomplishment(s), if any. IMPROVED METHODS OF PROTEIN PRODUCTION BY RHIZOPUS - performed under NP 306, Quality and Utilization of Agricultural Products. Rhizopus is recognized as a robust organism that is routinely used for industrial enzyme production (e.g., glucoamylase, lipase, peptidase), but its use is limited by the enzymes that the organism naturally produces. We continued our development of expression vectors to allow secretion of important proteins with Rhizopus. Furthermore, these vectors contain binding sites that will be of potential benefit for purification of the final protein. The application of this technology is expected to have direct benefits to numerous industries involved in production of enzymes, including those involved in biomass conversion. 5. Describe the major accomplishments to date and their predicted or actual impact. A major thrust of this project has been to use molecular biology techniques for the development of microbial strains with improved lactic acid production. We have isolated several genes from Rhizopus that are associated with lactic acid formation and more recently have been collaborating in the sequencing of Rhizopus genome. Numerous methods have been developed to introduce genetically modified genes back into the fungal host in order to decrease production of unwanted byproducts and increase accumulation of lactic acid. Each modification of a particular enzymatic conversion step resulted in varying degrees of improvement for fermentations using this fungus. This work has been so successful that it led to a two Cooperative Research and Development Agreements (CRADA) with a major industrial partner over the last seven years. We have also begun to utilize some of these molecular techniques to investigate the ability to express proteins of interest in Rhizopus. This work has been very promising and also resulted in the formation of a CRADA with a midwestern biotechnology company to express important industrial enzymes. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? We have had a CRADA for the last seven years with a major industrial partner so that they may assist in our efforts to increase lactic production in the fungus Rhizopus. Strains modified for improved synthesis of lactic acid were routinely transferred as part of this CRADA. The numerous techniques developed by our laboratory served as a foundation to allow the formation of another CRADA with a major midwestern biotechnology company to develop improved methods of enzyme synthesis using R. oryzae. Several of our expression systems have been tested with various genes from the CRADA partner. Ultimately, the farmer and consumer will benefit from all of this research, since it relies on renewable agricultural materials for the final product. Other scientists also benefit from this work, since we often assist other laboratories by providing strains, DNA, and knowledge from our research.

Impacts
(N/A)

Publications

  • Mertens, J.A., Skory, C.D. 2004. Discovery and characterization of a second glucoamylase gene in rhizopus oryzae [abstract]. Society of Industrial Microbiology. p. 96.
  • Skory, C.D. 2005. Inhibition of non-homologous end joining and integration of DNA with transformation of Rhizopus oryzae. Molecular Genetics and Genomics. 274(4):373-83.
  • Gorsich, S.W., Dien, B.S., Nichols, N.N., Slininger, P.J., Liu, Z., Skory, C.D. 2005. Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPEL, and TKL1 in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 71(3):339-349.
  • Mertens, J.A., Skory, C.D. 2005. Development of plasmids for expression of heterologous proteins in Rhizopus oryzae [abstract]. Society of Industrial Microbiology. p. 100.
  • Mertens, J.A., Skory, C.D., Ibrahim, A.S. 2006. Plasmids for expression of heterologous proteins in Rhizopus oryzae. Archives of Microbiology. 186:41- 50.


Progress 10/01/04 to 09/30/05

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Fossil fuels are by far the most exploited natural resource used today. The whole infrastructure of modern society is built upon oil, which has a limited reserve. Petroleum serves as the foundation for not only most of the USA's energy needs, but also for many products, such as solvents, organic acids, and numerous polymers and plastics. In 2004, the USA consumed about 5.7 billions of barrels of petroleum with approximately 2- 5% being used for the production of plastics and solvents (Department of Energy (DOE) Petroleum Supply Annual 2004). Although many benefits to society are derived from the use of petroleum, potential problems, in the form of litter, landfill depletion, environmental pollution, global warming, and increased atmospheric carbon dioxide are associated with its use. Means to help alleviate these problems include the development of viable technologies for the production of industrial chemicals using renewable feedstocks (e.g., starch or cellulosic biomass) and the development of more efficient fermentation processes. Finding new uses for agricultural crops and improving the efficiency of how we utilize them is imperative for competing in today's global market. The broad objective of the CRIS Work Unit (CWU) is to develop improved technologies and novel systems for increasing yields and reducing costs of converting agricultural commodities/residues to value-added products (e.g., biofuels and organic acids) through enzyme and fermentation technology. The CWU concentrates primarily on studying the genetic control mechanisms for production of several metabolites and enzymes by the filamentous fungus Rhizopus oryzae. This fungus is used for large- scale manufacture of lactic acid. However, problems exist with fermentations because of synthesis of several unwanted byproducts that must be separated from the final product and decrease the overall yield of lactic acid. This fungus is also recognized as a robust organism that is routinely used for industrial enzyme production (e.g., glucoamylase, lipase, peptidase). Currently, its use is limited by the enzymes that the organism naturally produces. We have developed technologies that can now be used to genetically manipulate this fungus so that a host of useful proteins can be produced. This research contributes to the objectives of NP 306 New Processes, New Uses and Value-Added Foods and Biobased Products, component Biobased Non- Food Carbohydrate and Protein Processing. Corn and its processing byproducts (e.g., corn stover and corn fiber) will initially serve as the primary agricultural crop for this research, although technology is applicable to alternative crops. Customers of this work include: producers/consumers of lactic acid or its end-products (e.g., polylactic acid (PLA) plastic); industries requiring enzymes (e.g., biofuels, biomass conversion, food and feed, specialty chemicals); corn processors; new markets for agricultural commodities; agricultural community; scientific community with interests in fungal metabolic engineering; and general public. Cooperative Research and Development Agreements (CRADA) with appropriate companies are currently used to transfer products of this research. Much of the research focuses on the production of lactic acid because of the potential market growth of the biodegradable plastic, polylactic acid, and solvent ethyl lactate. Domestic production of lactic acid is greater than 50,000 tons/yr and will likely increase exponentially if the growth for PLA and ethyl lactate develops as expected. However, fermentation efficiency must be improved to ensure the economic feasibility of the anticipated market expansion and to ensure that the U. S. maintains its leadership role. Additionally, considerable effort is being directed towards new technologies for improved enzyme production, which currently are estimated to have annual sales in excess of $1 billion. The ability to genetically improve the production of these enzymes, as well as other novel recombinant enzymes (e.g., cellulase, xylanase), is expected to have significant implications. Furthermore, the genetic knowledge gained from this work can easily be adapted to aid research with numerous other fungi that have industrial value. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Test feasibility of using Rhizopus oryzae for expression of desired proteins. Continue to modify expression of various genes involved in the synthesis of lactic acid and various unwanted byproducts. Year 2 (FY 2005) Begin study of protein secretion pathways for Rhizopus. Continue study of synthesis pathways for unwanted products formed during lactic acid production. Develop improved methods of integration and gene-knockout. Year 3 (FY 2006) Design improved methods of protein secretion. Apply technology of gene integration and gene-knockout to ameliorate production of unwanted by-products. Initiate development of biochemical and analytic tools for monitoring the metabolic state of Rhizopus during fermentation. Initiate work to investigate inhibitors of lactic acid production in Rhizopus. Year 4 (FY 2007) Utilize new analytical techniques to study modified strains and identify new targets for further improvements in lactic acid production and protein synthesis. Begin testing of pilot and large scale fermentations of modified strains. Identify targets to overcome inhibition of lactic acid production. Year 5 (FY 2008) Test improved protein production systems using several enzymes with industrial interest. Study methods to improve product stability during lactic acid production. Utilize technology of gene modification to improve lactic acid production in the presence of inhibitors. Continue pilot and large scale testing of modified strains. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Begin study of protein secretion pathways for Rhizopus. Milestone Fully Met 2. Continue study of synthesis pathways for unwanted products formed during lactic acid production. Milestone Fully Met 3. Develop improved methods of integration and gene-knockout. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006 Design improved methods of protein secretion. Apply technology of gene integration and gene-knockout to ameliorate production of unwanted by-products. Initiate development of biochemical and analytic tools for monitoring the metabolic state of Rhizopus during fermentation. Initiate work to investigate inhibitors of lactic acid production in Rhizopus. FY 2007 Utilize new analytical techniques to study modified strains and identify new targets for further improvements in lactic acid production and protein synthesis. Begin testing of pilot and large scale fermentations of modified strains. Identify targets to overcome inhibition of lactic acid production. FY 2008 Test improved protein production systems using several enzymes with industrial interest. Study methods to improve product stability during lactic acid production. Utilize technology of gene modification to improve lactic acid production in the presence of inhibitors. Continue pilot and large scale testing of modified strains. 4a What was the single most significant accomplishment this past year? MOLECULAR BIOLOGY METHODS. Research with Rhizopus has been hampered by a lack of molecular techniques for the genetic manipulation of this fungus, thereby impeding the ability to study the organism and exploit its valuable traits. Furthermore, many other related fungi of industrial interest share this same dilemma mainly due to a lack of knowledge regarding the mechanisms that control the replication and repair of DNA. We have developed novel methods of introducing DNA into Rhizopus in a controlled, targeted, and selectable manner that represents a significant advance in Rhizopus genetics. The results of this study will allow new strategies to be developed that can exploit this discovery and allow more rapid progression of the industrial utilization of this valuable organism. 4b List other significant accomplishments, if any. PROTEIN EXPRESSION. Rhizopus is recognized as a robust organism that is routinely used for industrial enzyme production (e.g., glucoamylase, lipase, peptidase), but its use is limited by the enzymes that the organism naturally produces. We have developed numerous vectors that are being used to study the expression of several genes of interest. This work also allows us to more closely examine the ability to secrete important proteins with Rhizopus. The application of this technology is expected to have direct benefits to numerous industries involved in production of enzymes, including those involved in biomass conversion. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A major thrust of this project has been to use molecular biology techniques for the development of microbial strains with improved lactic acid production. We have isolated several genes from Rhizopus that are associated with lactic acid formation. Methods were then developed to introduce genetically modified genes back into the fungal host in order to decrease production of unwanted byproducts and increase accumulation of lactic acid. Each modification of a particular enzymatic conversion step resulted in varying degrees of improvement for fermentations using this fungus. This work was so successful that it led to a Cooperative Research and Development Agreement (CRADA) with a major industrial partner, which has been ongoing for the last six years. Furthermore, we have also begun to utilize some of these molecular techniques to investigate the ability to express proteins of interest in Rhizopus. This work has been very promising and also resulted in the formation of a CRADA with a midwestern biotechnology company to express important industrial enzymes. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? We have had a CRADA for the last six years with a major industrial partner so that they may assist in our efforts to increase lactic production in the fungus Rhizopus. Strains modified for improved synthesis of lactic acid are routinely transferred as part of this CRADA. Technology resulting from this project has been licensed and is being used routinely by the CRADA partner. The numerous techniques developed by our laboratory served as a foundation to allow the formation of another CRADA with a major midwestern biotechnology company to develop improved methods of enzyme synthesis using R. oryzae. Several of our expression systems have been tested with various genes from the CRADA partner. Ultimately, the farmer and consumer will benefit from all of this research, since it relies on renewable agricultural materials for the final product. Other scientists also benefit from this work, since we often assist other laboratories by providing strains, DNA, and knowledge from our research.

Impacts
(N/A)

Publications

  • Dien, B.S., Iten, L.B., Skory, C.D. 2005. Converting herbaceous energy crops to bioethanol: a review with emphasis on pretreatment processes. In: Hou, C.T., editor. Handbook of Industrial Biocatalysis. Chapter 23. Boca Raton, FL: Taylor & Francis Group. p. 1-11.
  • Dien, B.S., Whitehead, T.R., Nichols, N.N., Skory, C.D., Cotta, M.A. 2004. Recombinant biocatalysts for converting sugar mixtures to lactic acid [abstract]. Great Lakes Regional American Chemical Society Symposium. Paper No. 40.