Progress 07/01/19 to 05/31/20

Outputs
Target Audience: Primordial Genetics' target audience for this project is the ethanol industry, the yeast companies that supply it, and the ethanol industry's downstream customer base that uses ethanol dry mill co-products for animal feed and represents an important part of the complex supply chain for meat production. Our direct target customers include companies that supply yeast to ethanol producers. We have been in conversations with companies including Lallemand, Lesaffre (Leaf), Novozymes and ADM. All four companies expressed interest in the project at an early stage and three confirmed their interest with a letter of support for our Phase II application. We continue to provide all interested parties with up to date information regarding progress in the project including a summary of our Phase I results and plans for Phase II. We have also engaged in extensive conversations with process technology companies (ICM, Fluid Quip Process Technologies) and ethanol producers (Flint Hills Resources, ADM) who confirmed the trends for dry mill modifications to produce higher value animal feed ingredients. Finally, we have had numerous conversations with animal nutritionists, animal nutrition companies and animal producers (e.g. Perdue Farms, Alltech, Archer Daniel Midlands, Devenish Nutrition, Tyson Foods, Smithfield Foods), and have confirmed the view that methionine demand will continue to grow. Those conversations have also confirmed that there is an unmet need for a cost-effective biological methionine supply, preferably one that is biologically produced in North America. Changes/Problems: The stated goal for this project was to develop at least one candidate strain that accumulates at least 2% of its total protein content as methionine (2x higher than the parent strain) under ethanol fermentation conditions. While we obtained at least 2x improvements in total methionine (bound in protein and free) under aerobic conditions, we do not believe the majority of this excess methionine to be accumulating in protein, but believe a large majority of it exists as free methionine. This is not a problem as accumulation of both free and bound methionine will be included in the final DDGS product. The fluorescent E. coli indicator strain used for our methionine bioassay was deficient in cystathionine γ-synthase (metB) which catalyzes the conversion of O-succinylhomoserine to cystathionine. Cystathionine lies two enzymatic steps upstream of methionine in the biosynthetic pathway. Therefore, in principle, the bioassay measures the combined levels of methionine, cystathionine and homocysteine. While high levels of cystathionine and homocysteine are potentially toxic and unlikely to accumulate in cells in appreciable amounts, the lack of specificity of this assay for methionine is the reason for the use of confirmatory assays which included LC-MS and a commercially available fluorescence-based methionine assay (Abcam plc, Cambridge, MA) in Phase I. We have proposed improvements to this bioassay assay in our Phase II proposal. UV mutagenesis of the diploid Ethanol Red strain did not readily yield stable high-methionine mutants. Initial screening in our bioassay resulted in 33 mutants showing 1.2-2x improvements in methionine levels over wild type. The increased methionine observed in the initial screening of UV isolates was not as robust upon additional rounds of testing and may be due epigenetic effects and/or issues with trying to mutate a diploid strain. Unlike the UV mutants, most of our Function Generator™ genes maintained their increased methionine levels and were the focus of the majority of the Phase I work. We initially proposed to test at least 25 unique strains identified from the methionine analog screening that would be grown in shake flask ethanol fermentations and methionine levels tested in our bioassay. Samples were to be collected after complete fermentation and analyzed for ethanol, yeast dry cell weight, protein and methionine content (in yeast pellet and culture media). We did not perform these larger scale shake flask fermentations as we decided to test far more than 25 unique strains for improved methionine content. We opted for small scale aerobic cultures and screened over 500 strains for increased methionine levels in order to improve our chances of finding unique genetic solutions that increased levels of methionine. Due to the large number of samples tested, we did not analyze ethanol, yeast dry cell weight or protein in these samples. This analysis will be done in Phase II using process-relevant fermentation systems. We observed our goal of 2x improvements in methionine levels with single Function Generator™ genes, and hoped combinations of Function Generator™ genes would lead to even greater improvements. Combinations of Function Generator™ genes gave only modest increases in methionine levels compared to single Function Generator™ genes. Rather than spend more time on combinatorial screening that would need to be redone at a later stage under anaerobic growth conditions, we decided to begin investigating other fundamental ways of boosting yeast methionine content not mentioned in the initial proposal. We initiated additional lines of experimentation that will be continued in Phase II which involved testing of active Function Generator™ genes under anaerobic growth conditions, expression of methionine-rich proteins as methionine sinks, and testing of S-adenosylmethionine synthetase (SAMS) knockout mutants. We felt that these efforts represented a beter use of our time and the project funds and would lay a more solid foundation for additional work to be done in Phase II. We have met our goals for phase I proof of concept, but have adjusted our initial Phase II target for increase in methionine levels from 4x to 10x. This was done as a consequence of our own internal techno-economic analysis of this opportunity which indicated that raising methionine levels beyond 4x would allow us to generate far more value, and also as a result of industry feedback. Yeast suppliers that we have been in contact with have provided input on what concentrations of methionine in spent yeast from ethanol fermentation is required to make the HMY value proposition commercially viable. The consensus among several companies we have talked to is that we need to achieve 10x higher methionine levels relative to the methionine content of currently used yeast strains with minimal impact on ethanol productivity. We are confident that combinations of Function Generator™ genes, metabolic engineering, and methionine sinks we initiated in Phase I and plan to continue in Phase II will allow us to reach this goal. What opportunities for training and professional development has the project provided?Daniel Olson PhD was the scientist undertaking the majority of the experimental work of this Phase I project. During the scope of this project, Dr. Olson took over the project leadership from Dr. Justin Stege, the original PD, and assumed the responsibilities of organizing the experimental work and reporting the results of the project to the appropriate government agencies. In addition, Dr. Olson has now been involved in the writing and submission of the Phase II SBIR proposal associated with this Phase I project. On the strength of his professional growth in the course of the project Dr. Olson was promoted by Primordial Genetics to Senior Scientist. The professional development Dr. Olson received during the scope of this project were under the guidance of Primordial Genetics' former Vice President of R&D Justin Stege PhD and President Helge Zieler PhD. How have the results been disseminated to communities of interest?We have been in conversations with yeast companies including Lallemand, Lesaffre (Leaf), Novozymes and ADM. All four companies expressed interest in the project at an early stage and were provided with a Phase I summary at the end of the project. Three companies confirmed their continued interest in development of high-methionine yeast with a letter of support for our Phase II application. In addition to yeast companies, we have we have engaged in numerous conversations with animal nutritionists, animal nutrition companies and animal producers (e.g. Perdue Farms, Alltech, Archer Daniel Midlands, Devenish Nutrition, Tyson Foods, Smithfield Foods), process technology companies (ICM, Fluid Quip Process Technologies) and ethanol producers (Flint Hills Resources, ADM). These contacts have allowed us to educate the animal feed and meat production industries about our efforts and receive their feedback on our project goals and the needs of the target industries. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Increasing world demand for animal protein is driving market growth for amino acids as feed supplements. Methionine commands the highest market value of all amino acids and has a worldwide annual value of approximately $5 Billion with an expected 5-7% annual growth over the next decade. Methionine is produced by chemical synthesis, and previous attempts at biological production have been unsuccessful in commercializing an economical bio-methionine process. The goal of this project is to develop high methionine yeast (HMY) strains to improve the nutritional content of yeast-based co-products. Using an HMY yeast strain in ethanol production would allow ethanol plants to produce an enhanced protein product with a higher market value and generate increased revenues. The Phase I project was highly successful as we discovered 14 independent genetic solutions giving at least a 50% increase in yeast methionine content with some more than doubling methionine. We demonstrated the feasibility of using Primordial Genetics' technology to improve the methionine content of an industrial yeast strain. Results Objective 1: Screen Primordial Genetics' yeast Function Generator library in a commercial yeast strain for novel genes that boost methionine production. The previously constructed S. cerevisiae FG library, encoding 3.1x107 compound genes, was introduced into a variant of the commercial ethanol yeast strain Ethanol Red and screened using three methionine analogs that provided mild to strong growth inhibition to identify resistant strains that are likely to overproduce methionine. We isolated and confirmed 524 clones derived from the population of FG library transformants that confer the ability to grown on methionine analogs Objective 2: Independently screen mutagenized yeast populations that boost yeast methionine production. As a source of additional genetic solutions and to allow a technology comparison with FG, we mutagenized yeast by UV irradiation. UV-mutagenized yeast cells were screened for growth on methionine analogs and 80 clones isolated capable of overcoming the growth inhibition of the methionine analogs. Objective 3: Discover at least 10 unique genetic solutions that increase methionine accumulation in shake flasks by 50%. To confirm increased methionine levels in strains, we used a previously developed methionine bioassay in which fluorescence of the bacterial culture reflects the amount of methionine present in a yeast cell extract. We compared this bioassay to a quantitative biochemical method using liquid chromatography coupled to mass spectrometry (LC-MS) and found excellent correlation between methionine levels measured with the two methods. The bioassay was used to measure methionine levels in 524 FG isolates representing at least 250 unique FG genes. 68 isolates showed increases in fluorescence correlating to increased methionine ranging from 1.2-2X improvements over wild type. Plasmids were rescued from all 524 FG isolates and FG genes sequenced. We classified the various open reading frames from the FG genes into 10 broad categories of methionine biosynthesis, methionine catabolism, amino acid metabolism, protein synthesis, proteasome, DNA repair and recombination, amino acid uptake, intracellular protein transport, primary metabolism, and unknown function. Eighty UV mutant isolates were screened in a similar manner. In contrast to the FG genes, the phenotypes of the UV mutants were not stable and the UV mutants were not characterized further. Objective 4: Demonstrate the potential to combine beneficial traits in the same strain to obtain a methionine content 2x higher than the parent strain. Even though the Phase I goal of 2x increases in methionine content were already met by several of the FG genes discovered in Objective 1 and 3, we postulated that combinations of FG genes may result in additional methionine gains. We constructed a library containing 12,100 combinations of FG gene pairs derived from 110 unique FG genes identified in our first round of screening. The library was transformed into the yeast strain and re-screened on methionine analogs in liquid culture. The best clones were isolated, validated and shown to confer 1.2-2x higher methionine content, similar to the improvements seen with single FG genes. The best FG genes and gene combinations from Objective 3 and 4 were tested in replicate samples to establish accurate measurements of the increase in methionine levels. We used a commercially available fluorescence-based methionine assay to confirm the measurements obtained with the bioassay. Both assays showed significant increases in methionine content: up to 2.7x above controls with the bioassay and >20x above controls using the commercial assay. Eight genes were confirmed to have >50% increase in methionine levels as measured with the bioassay data and 14 based on the commercial assay. We consider these discoveries as satisfying the main milestone of 10 or more genetic solutions that we set ourselves for the Phase I project. Additional work completed Testing of active FG genes under anaerobic growth conditions. To demonstrate the applicability of the FG genes isolated to increase methionine under process-relevant conditions, we set up small-scale anaerobic experiments using three unique FG genes with varying degrees of methionine improvement isolated under Objectives 3 and 4. Bioassay results from anaerobic conditions resulted in methionine improvements of 1.2x, 1.2x, and 1.3x, respectively. Although the improvements are less then what was observed under aerobic conditions, these data are promising and suggest the genetic solutions discovered may be transferable to anaerobic fermentations. Expression of methionine-rich proteins as methionine sinks. We postulated that creating a methionine sink would induce methionine production and help sequester excess methionine. We surveyed methionine-rich proteins expressed by yeast and other organisms and selected 6 of these for synthesis and expression in yeast. Expression of 2 of the 6 proteins was detectable by Western blot but weak. We concluded that it will take substantially higher effort to create a methionine sink. However, having identified two methionine-rich proteins that are expressed in S. cerevisiae is a good starting point for continuing this work in Phase II. Testing of S-Adenosylmethionine synthase (SAMS) knockout mutants. S-adenosylmethionine synthetase (SAMS) is the rate-limiting enzyme for methionine catabolism, and knockouts of either of the two yeast genes encoding isoforms of this enzyme (SAM1 and SAM2) have been shown to boost methionine levels. We obtained haploid knockout strains of the two SAM genes encoded by yeast and measured their methionine levels. Both mutants showed insignificant changes in methionine levels using our bioassay. We transformed both strains with several of our best FG genes isolated under Objective 3. The resulting transformants again showed only minor differences in methionine levels compared to the unmodified haploid control strain. However, the presence of the other isoform of the enzyme in both strains makes these negative results inconclusive. Because of the central role played by the SAMS enzymes in methionine breakdown, the SAM1 and SAM2 genes will continue to be targets for genetic modifications in Phase II. Conclusions The Phase I project was highly successful as we discovered multiple independent genetic solutions giving at least a 50% increase in methionine content with some more than doubling methionine compared to controls. We demonstrated the feasibility of using Primordial Genetics technology to improve the methionine content of an industrial yeast strain. Phase II will build upon the assays and genetics developed in Phase I in order to deliver the levels of improvement in yeast methionine content that will be required for development of a commercial high-methionine ethanol yeast strain.

Publications

Progress 07/01/19 to 05/30/20

Outputs
Target Audience: Primordial Genetics' target audience for this project is the ethanol industry, the yeast companies that supply it, and the ethanol industry's downstream customer base that uses ethanol dry mill co-products for animal feed and represents an important part of the complex supply chain for meat production. Our direct target customers include companies that supply yeast to ethanol producers. We have been in conversations with companies including Lallemand, Lesaffre (Leaf), Novozymes and ADM. All four companies expressed interest in the project at an early stage and three confirmed their interest with a letter of support for our Phase II application. We continue to provide all interested parties with up to date information regarding progress in the project including a summary of our Phase I results and plans for Phase II. We have also engaged in extensive conversations with process technology companies (ICM, Fluid Quip Process Technologies) and ethanol producers (Flint Hills Resources, ADM) who confirmed the trends for dry mill modifications to produce higher value animal feed ingredients. Finally, we have had numerous conversations with animal nutritionists, animal nutrition companies and animal producers (e.g. Perdue Farms, Alltech, Archer Daniel Midlands, Devenish Nutrition, Tyson Foods, Smithfield Foods), and have confirmed the view that methionine demand will continue to grow. Those conversations have also confirmed that there is an unmet need for a cost-effective biological methionine supply, preferably one that is biologically produced in North America. Changes/Problems: The stated goal for this project was to develop at least one candidate strain that accumulates at least 2% of its total protein content as methionine (2x higher than the parent strain) under ethanol fermentation conditions. While we obtained at least 2x improvements in total methionine (bound in protein and free) under aerobic conditions, we do not believe the majority of this excess methionine to be accumulating in protein, but believe a large majority of it exists as free methionine. This is not a problem as accumulation of both free and bound methionine will be included in the final DDGS product. The fluorescent E. coli indicator strain used for our methionine bioassay was deficient in cystathionine γ-synthase (metB) which catalyzes the conversion of O-succinylhomoserine to cystathionine. Cystathionine lies two enzymatic steps upstream of methionine in the biosynthetic pathway. Therefore, in principle, the bioassay measures the combined levels of methionine, cystathionine and homocysteine. While high levels of cystathionine and homocysteine are potentially toxic and unlikely to accumulate in cells in appreciable amounts, the lack of specificity of this assay for methionine is the reason for the use of confirmatory assays which included LC-MS and a commercially available fluorescence-based methionine assay (Abcam plc, Cambridge, MA) in Phase I. We have proposed improvements to this bioassay assay in our Phase II proposal. UV mutagenesis of the diploid Ethanol Red strain did not readily yield stable high-methionine mutants. Initial screening in our bioassay resulted in 33 mutants showing 1.2-2x improvements in methionine levels over wild type. The increased methionine observed in the initial screening of UV isolates was not as robust upon additional rounds of testing and may be due epigenetic effects and/or issues with trying to mutate a diploid strain. Unlike the UV mutants, most of our Function Generator™ genes maintained their increased methionine levels and were the focus of the majority of the Phase I work. We initially proposed to test at least 25 unique strains identified from the methionine analog screening that would be grown in shake flask ethanol fermentations and methionine levels tested in our bioassay. Samples were to be collected after complete fermentation and analyzed for ethanol, yeast dry cell weight, protein and methionine content (in yeast pellet and culture media). We did not perform these larger scale shake flask fermentations as we decided to test far more than 25 unique strains for improved methionine content. We opted for small scale aerobic cultures and screened over 500 strains for increased methionine levels in order to improve our chances of finding unique genetic solutions that increased levels of methionine. Due to the large number of samples tested, we did not analyze ethanol, yeast dry cell weight or protein in these samples. This analysis will be done in Phase II using process-relevant fermentation systems. We observed our goal of 2x improvements in methionine levels with single Function Generator™ genes, and hoped combinations of Function Generator™ genes would lead to even greater improvements. Combinations of Function Generator™ genes gave only modest increases in methionine levels compared to single Function Generator™ genes. Rather than spend more time on combinatorial screening that would need to be redone at a later stage under anaerobic growth conditions, we decided to begin investigating other fundamental ways of boosting yeast methionine content not mentioned in the initial proposal. We initiated additional lines of experimentation that will be continued in Phase II which involved testing of active Function Generator™ genes under anaerobic growth conditions, expression of methionine-rich proteins as methionine sinks, and testing of S-adenosylmethionine synthetase (SAMS) knockout mutants. We felt that these efforts represented a beter use of our time and the project funds and would lay a more solid foundation for additional work to be done in Phase II. We have met our goals for phase I proof of concept, but have adjusted our initial Phase II target for increase in methionine levels from 4x to 10x. This was done as a consequence of our own internal techno-economic analysis of this opportunity which indicated that raising methionine levels beyond 4x would allow us to generate far more value, and also as a result of industry feedback. Yeast suppliers that we have been in contact with have provided input on what concentrations of methionine in spent yeast from ethanol fermentation is required to make the HMY value proposition commercially viable. The consensus among several companies we have talked to is that we need to achieve 10x higher methionine levels relative to the methionine content of currently used yeast strains with minimal impact on ethanol productivity. We are confident that combinations of Function Generator™ genes, metabolic engineering, and methionine sinks we initiated in Phase I and plan to continue in Phase II will allow us to reach this goal. What opportunities for training and professional development has the project provided?Daniel Olson PhD was the scientist undertaking the majority of the experimental work of this Phase I project. During the scope of this project, Dr. Olson took over the project leadership from Dr. Justin Stege, the original PD, and assumed the responsibilities of organizing the experimental work and reporting the results of the project to the appropriate government agencies. In addition, Dr. Olson has now been involved in the writing and submission of the Phase II SBIR proposal associated with this Phase I project. On the strength of his professional growth in the course of the project Dr. Olson was promoted by Primordial Genetics to Senior Scientist. The professional development Dr. Olson received during the scope of this project were under the guidance of Primordial Genetics' former Vice President of R&D Justin Stege PhD and President Helge Zieler PhD. How have the results been disseminated to communities of interest?We have been in conversations with yeast companies including Lallemand, Lesaffre (Leaf), Novozymes and ADM. All four companies expressed interest in the project at an early stage and were provided with a Phase I summary at the end of the project. Three companies confirmed their continued interest in development of high-methionine yeast with a letter of support for our Phase II application. In addition to yeast companies, we have we have engaged in numerous conversations with animal nutritionists, animal nutrition companies and animal producers (e.g. Perdue Farms, Alltech, Archer Daniel Midlands, Devenish Nutrition, Tyson Foods, Smithfield Foods), process technology companies (ICM, Fluid Quip Process Technologies) and ethanol producers (Flint Hills Resources, ADM). These contacts have allowed us to educate the animal feed and meat production industries about our efforts and receive their feedback on our project goals and the needs of the target industries. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Increasing world demand for animal protein is driving market growth for amino acids as feed supplements. Methionine commands the highest market value of all amino acids and has a worldwide annual value of approximately $5 Billion with an expected 5-7% annual growth over the next decade. Methionine is produced by chemical synthesis, and previous attempts at biological production have been unsuccessful in commercializing an economical bio-methionine process. The goal of this project is to develop high methionine yeast (HMY) strains to improve the nutritional content of yeast-based co-products. Using an HMY yeast strain in ethanol production would allow ethanol plants to produce an enhanced protein product with a higher market value and generate increased revenues. The Phase I project was highly successful as we discovered 14 independent genetic solutions giving at least a 50% increase in yeast methionine content with some more than doubling methionine. We demonstrated the feasibility of using Primordial Genetics' technology to improve the methionine content of an industrial yeast strain. Results Objective 1: Screen Primordial Genetics' yeast Function Generator library in a commercial yeast strain for novel genes that boost methionine production. The previously constructed S. cerevisiae FG library, encoding 3.1x107 compound genes, was introduced into a variant of the commercial ethanol yeast strain Ethanol Red and screened using three methionine analogs that provided mild to strong growth inhibition to identify resistant strains that are likely to overproduce methionine. We isolated and confirmed 524 clones derived from the population of FG library transformants that confer the ability to grown on methionine analogs Objective 2: Independently screen mutagenized yeast populations that boost yeast methionine production. As a source of additional genetic solutions and to allow a technology comparison with FG, we mutagenized yeast by UV irradiation. UV-mutagenized yeast cells were screened for growth on methionine analogs and 80 clones isolated capable of overcoming the growth inhibition of the methionine analogs. Objective 3: Discover at least 10 unique genetic solutions that increase methionine accumulation in shake flasks by 50%. To confirm increased methionine levels in strains, we used a previously developed methionine bioassay in which fluorescence of the bacterial culture reflects the amount of methionine present in a yeast cell extract. We compared this bioassay to a quantitative biochemical method using liquid chromatography coupled to mass spectrometry (LC-MS) and found excellent correlation between methionine levels measured with the two methods. The bioassay was used to measure methionine levels in 524 FG isolates representing at least 250 unique FG genes. 68 isolates showed increases in fluorescence correlating to increased methionine ranging from 1.2-2X improvements over wild type. Plasmids were rescued from all 524 FG isolates and FG genes sequenced. We classified the various open reading frames from the FG genes into 10 broad categories of methionine biosynthesis, methionine catabolism, amino acid metabolism, protein synthesis, proteasome, DNA repair and recombination, amino acid uptake, intracellular protein transport, primary metabolism, and unknown function. Eighty UV mutant isolates were screened in a similar manner. In contrast to the FG genes, the phenotypes of the UV mutants were not stable and the UV mutants were not characterized further. Objective 4: Demonstrate the potential to combine beneficial traits in the same strain to obtain a methionine content 2x higher than the parent strain. Even though the Phase I goal of 2x increases in methionine content were already met by several of the FG genes discovered in Objective 1 and 3, we postulated that combinations of FG genes may result in additional methionine gains. We constructed a library containing 12,100 combinations of FG gene pairs derived from 110 unique FG genes identified in our first round of screening. The library was transformed into the yeast strain and re-screened on methionine analogs in liquid culture. The best clones were isolated, validated and shown to confer 1.2-2x higher methionine content, similar to the improvements seen with single FG genes. The best FG genes and gene combinations from Objective 3 and 4 were tested in replicate samples to establish accurate measurements of the increase in methionine levels. We used a commercially available fluorescence-based methionine assay to confirm the measurements obtained with the bioassay. Both assays showed significant increases in methionine content: up to 2.7x above controls with the bioassay and >20x above controls using the commercial assay. Eight genes were confirmed to have >50% increase in methionine levels as measured with the bioassay data and 14 based on the commercial assay. We consider these discoveries as satisfying the main milestone of 10 or more genetic solutions that we set ourselves for the Phase I project. Additional work completed Testing of active FG genes under anaerobic growth conditions. To demonstrate the applicability of the FG genes isolated to increase methionine under process-relevant conditions, we set up small-scale anaerobic experiments using three unique FG genes with varying degrees of methionine improvement isolated under Objectives 3 and 4. Bioassay results from anaerobic conditions resulted in methionine improvements of 1.2x, 1.2x, and 1.3x, respectively. Although the improvements are less then what was observed under aerobic conditions, these data are promising and suggest the genetic solutions discovered may be transferable to anaerobic fermentations. Expression of methionine-rich proteins as methionine sinks. We postulated that creating a methionine sink would induce methionine production and help sequester excess methionine. We surveyed methionine-rich proteins expressed by yeast and other organisms and selected 6 of these for synthesis and expression in yeast. Expression of 2 of the 6 proteins was detectable by Western blot but weak. We concluded that it will take substantially higher effort to create a methionine sink. However, having identified two methionine-rich proteins that are expressed in S. cerevisiae is a good starting point for continuing this work in Phase II. Testing of S-Adenosylmethionine synthase (SAMS) knockout mutants. S-adenosylmethionine synthetase (SAMS) is the rate-limiting enzyme for methionine catabolism, and knockouts of either of the two yeast genes encoding isoforms of this enzyme (SAM1 and SAM2) have been shown to boost methionine levels. We obtained haploid knockout strains of the two SAM genes encoded by yeast and measured their methionine levels. Both mutants showed insignificant changes in methionine levels using our bioassay. We transformed both strains with several of our best FG genes isolated under Objective 3. The resulting transformants again showed only minor differences in methionine levels compared to the unmodified haploid control strain. However, the presence of the other isoform of the enzyme in both strains makes these negative results inconclusive. Because of the central role played by the SAMS enzymes in methionine breakdown, the SAM1 and SAM2 genes will continue to be targets for genetic modifications in Phase II. Conclusions The Phase I project was highly successful as we discovered multiple independent genetic solutions giving at least a 50% increase in methionine content with some more than doubling methionine compared to controls. We demonstrated the feasibility of using Primordial Genetics technology to improve the methionine content of an industrial yeast strain. Phase II will build upon the assays and genetics developed in Phase I in order to deliver the levels of improvement in yeast methionine content that will be required for development of a commercial high-methionine ethanol yeast strain.

Publications