Progress 11/15/05 to 11/14/08
Outputs
OUTPUTS: This USDA-NRI proposal, co-funded by NSF, focused on enzyme interactions among the key enzymes in the phenylpropanoid pathway. We have made significant advances in the field with many interesting discoveries. During the funding period, 24 papers (including book chapters) have been published (including "in press") from my lab. In addition, we were invited to give talks at 11 meetings or universities. We submitted 15 posters/abstracts at different conferences and symposia. With the data generated by this funding, we were able to obtain additional federal and local grants. For example, we were recently awarded a DOE Energy Frontier Research Center Grant in the amount of $15 million for 5 years. Enzyme organizations and their regulation of pathway flux are essential aspects of metabolic research in a post-genome era. Compared to the wealth of information regarding the transcriptional regulations and metabolomics profiles of various pathways, little is known about the cellular mechanisms of these metabolic processes. Exactly how cells direct the flow of common substrates towards each sub-branch of a pathway under specific conditions is still not clear. Extensive studies have demonstrated that key natural product enzymes form macromolecular complexes or "metabolons" that channel metabolites into different pathways. These specific enzyme interactions are sometimes the main bottlenecks for metabolic engineering in plants. We have successfully reconstituted many parts of the flavonoid pathway in yeast by expressing key enzymes and supplying various substrates to the culture media. We demonstrated that the flow of flavonoid intermediates in yeast was influenced by specific enzyme associations. This in vivo yeast system thus provides us a unique platform for studying the flux of metabolites and interactions of enzymes. During the funding period, we focused on understanding how interactions between the key flavonoid enzymes direct the pathway flux towards a particular branch of a pathway. Information gained in mechanistic studies from yeast allowed us to design better metabolic engineering approaches in microbes, mammalian cells, and plants. Engineering of plant flavonoid profiles led us to investigate the functions of these flavonoids in defense responses and in legume-rhizobium interactions. The following section are brief summaries of some discoveries. PARTICIPANTS: Oliver Yu, PD, Danforth Plant Science Center. Dr. Yechun Wang, 2007- current postdoc assistant. Miss Hui Li, visiting graduate student, Shanghai Jiaotong University, Shanghai, China. Dr. Senthil Subramanian, 2002- 2009 postdoc associate, currently assistant professor at South Dakota State University. Dr. Juan Zhang, 2005- 2008, postdoc assistant, currently Assistant Professor at Ludong University, Yantai, China. Dr. Coralie Halls, 2006- 2007 postdoc assistant, currently senior research fellow of Monsanto Co. Dr. Congbing Fang, 2006- 2007 visiting scientist, Professor, Anhui Agricultural University, Hefei, China. Dr. Un-Haing Cho, 2007- 2008 visiting scientist, Professor and Provost for Research at Changwon National University, Changwon, Korea. Amanda Smith, summer intern 2008, University of Missouri St Louis, currently lab technician at Washington University in St Louis. Kara Higgs, summer intern 2007, Ohio State University. Juan Jose Gutierrez, graduate student, Agronomy Department, University of Missouri-Columbia. (Graduate committee member, since 2006). Sarah Marie Sexton, graduate student, Agronomy Department, University of Missouri-Columbia. (Graduate committee member, since 2007), currently Research Scientist at Monsanto Company. Dr. Ahmed Elshehawi, 2007-2007, visiting scientist, Associate Professor from Alexandria University, Alexandria, Egypt. Dr. Yansheng Zhang, Postdoc 2005-2006, currently Research Fellow, Agriculture Research Counsel, Canada. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. Yeast platform: By expressing one or more phenylpropanoid enzymes in S. cerevisiae, the entire biosynthetic pathway can be established stepwise and functionally analyzed in yeast. Since most phenylpropanoid compounds have relatively hydrophobic phenol rings and are capable of passing through the cell membrane by diffusion, these transgenic yeast can be fed with various substrate and intermediates of the pathway, and the products of each enzymes can then be collected and quantified. We have used this system to study the gene expression, substrate specificity, enzyme kinetics, or reaction mechanisms of at least 14 enzymes of the pathway, including PAL, 4CL, HCT, CHS, type I and II CHIs, FNS, F3H, IFS, IDH, FLS, IOMT, C-GT, and TAL. 2. In vivo enzyme interactions in yeast: We found that defense-inducible type II CHI interacts with IFS enzyme to direct the flow of substrates towards isoflavone phytoalexin production in yeast. The IFS-CHI interaction can be detected by tandem affinity purification (TAP), immuno-co-precipitation (co-IP), and FRET assays. In yeast containing type II CHI, IFS, and a competing flavonoid enzyme FNS, the system produced 30% more isoflavones than flavones whenever CHI was involved. If CHI and IFS were physically linked together in a CHI::IFS fusion protein, the same increase in isoflavone production was observed. For the first time, we were able to show artificial channeling of CHI and IFS drives the flux of intermediate into one side of the pathway. When the same concept is tested in resveratrol biosynthesis, we were able to find one linker that joins 4CL and STS into an efficient fusion protein that produces almost 20-fold higher resveratrol in yeast than the co-expression of two free enzymes. 3. Metabolic engineering revealed cross-talks between different branches of flavonoid pathway: In addition to metabolic engineering in heterologous systems such as Arabidopsis, we also studied the connections between flavonoid and isoflavonoid biosynthesis in native species as well. When a synthetic Myb-like transcription factor was expressed in soybean seed, it activated many enzymes in both upstream phenylpropanoid pathway and the proanthocyanidin pathway, but not the enzymes in the isoflavone pathway, leading to brown coloration in seed coat, reduced genistein levels, and drastically higher daidzein levels. The results suggested isoflavone biosynthesis is linked to flavonoid biosynthesis in soybean. 4. Coordinated expression of phenylpropanoid genes in vascular tissues: To our surprise, legume flavonoid biosynthesis under stress conditions showed remarkable tissue-specific activation in the same pattern as lignin synthesis. Promoter-reporter analysis and in situ hybridization of the IFS and FNS genes in soybean and M. truncatula all pointed to specific gene activations in the vascular tissues, particularly in root xylem parenchyma cells. Just like in Arabidopsis, these flavonoid compounds play essential roles in auxin transport, and flavonoid regulation of auxin efflux carriers affects nodule and lateral root primordial development.
Publications
- Zhang Y, Liu ZH, Jia L, Peng ZL, Jaworski J, Wang XM, Jez J., Chen F, Yu O (2006) Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and mammalian cells. Journal of American Chemistry Society. 128: 13030-13031. Featured in Chemical and Engineering News, 84 (Oct. 2, 2006): page 43.
- Ralston L, Yu O. (2006) Metabolons involving plant cytochrome P450s. Phytochemistry Review. 5: 459-472.
- Yu O. (2006) Metabolic engineering of the plant phenylpropanoid pathway. Encyclopedia of Plant and Crop Science, Dekker Publishing. DOI: 10.1081/E-EPCS-120010589.
- Yu O, Matsuno M, Subramanian S. (2006) Flavonoids in flowers: Genetics and Biochemistry. In Floriculture, ornamental and plant biotechnology: Advances and topical issues (1st Edition), Jaime A Teixeira da Silva (ed.), pp. 283-293.
- Ralston L, Subramanian S, Matsuno M, Yu O. (2005) Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast (Saccharomyces cerevisiae) using soybean type I and type II chalcone isomerases. Plant Physiology 137: 1375-1388.
- Subramanian S, Graham ML, Yu O, Graham T. (2005) Silencing of soybean isoflavone synthase through an RNAi approach leads to silencing in non-transformed tissue and to enhanced susceptibility to Phytophthora sojae. Plant Physiology 137: 1345-1353.
- Yu O, McGonigle B. (2005) Metabolic engineering of isoflavone biosynthesis. Advances in Agronomy 86: 147-189.
- Invited Talks from (2005 to 2009):
- Auxin-related microRNAs are essential for nodule and lateral root development. World Soybean Research Conference VIII, Beijing, China, August, 2009.
- Genetic regulation and metabolic engineering of isoflavone biosynthesis. American Chemistry Society (ACS) Annual Meeting, Washington DC, July, 2009
- Flavonoids and auxin-related microRNAs play critical roles in nodule and lateral root development. Brookhaven National Laboratories, New York, May, 2009.
- Flavonoid functions in legume-rhizobium interactions. Banff Conference on Plant Metabolism 2008. Banff, Alberta, Canada. August 2008.
- Metabolic engineering of flavonoid biosynthesis revealed distinct functions of flavonoids in legume-rhizobium interactions. Soy 2008: Molecular and Cellular Biology of the Soybean. Indianapolis, July 2008.
- Novel and nodulation-regulated microRNAs in soybean roots. By Senthil Subramanian, Soy 2008: Molecular and Cellular Biology of the Soybean. Indianapolis, July 2008.
- Engineering isoflavone and resveratrol biosynthesis. Saint Louis Academy of Science. June 2008.
- Metabolic engineering of red wine compound resveratrol. The ASA-CSSA-SSSA International Annual Meetings, New Orleans, LA. November, 2007.
- Metabolic engineering of resveratrol biosynthesis. OECD Symposium Series (Frontiers in Transgenesis), St Louis, MO, September 2007.
- Zhang J, Subramanian S, Stacey G, Yu O (2009) Flavonoids and auxin-related microRNAs play critical roles in nodule and lateral root development. Model Legume Conferences, San Francisco, CA.
- Zhang J, Subramanian S, Stacey G, Yu O (2008) Metabolic engineering of flavonoid biosynthesis revealed distinct functions of flavonoids in legume-rhizobia interactions. Soy 2008, Molecular & Cellular Biology of the Soybean Conference. Indianapolis, IN.
- Zhang J, Subramanian S, Stacey G, Yu O (2008) Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti. Banff Conference on Plant Metabolism, Banff, Alberta, Canada.
- Graham MY, Sinden MR, Huge R, Subramanian, S, Yu O, St Martin S, Graham TL (2008) Silencing of defense-related genes reveals different mechanisms leading to race-specific resistance to Phytophthora in soybean. APS Centennial Meeting, Minnesapolis, MN.
- Zhang J, Subramanian S, Stacey G, Yu O (2008) Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti. Plant Biology 2008, Merida, Mexco.
- Function of flavonoids during nodule development. The 20th North American Nitrogen Fixation Conference, Milwaukee, WI. July 2007.
- Metabolic engineering of resveratrol biosynthesis. Gordon Research Conference (Plant Metabolic Engineering), Tilton, NH. July 2007.
- Communications to Congresses from 2005 to 2009:
- Zhang J, Subramanian S, Stacey G, Yu O (2009) Flavonoids and auxin-related microRNAs play critical roles in nodule and lateral root development. ASPB Annual Meeting, Honolulu, HI
- Subramanian S, Fu Y, Sunkar R, Barbazuk B, Zhu JK, Yu O (2008) Novel and nodulation-regulated microRNAs in soybean roots. Plant Biology 2008, Merida, Mexco.
- Schroeder AC, Kumaran S, Hicks LM, Cahoon RE, Yu O, Jez J (2008) Contributions of conserved serine and tyrosine residues to catalysis, ligand binding, and cofactor processing in the active site of tyrosine ammonia lyase. Plant Biology 2008, Merida, Mexco.
- Zhang J, Subramanian S, Yu O (2007) Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation. Plant Biology 2007, Chicago, IL. ASPB abstract.
- Subramanian S, Stacey G, Yu O (2006) Endogenous isoflavones are essential for the establishment of symbiosis between soybean and Bradyrhizobium japonicum. Plant Roots: From Genes to Form & Function. Columbia, MO (Abstract 13).
- Subramanian S, Stacey G, Yu O (2005) Isoflavone's function in soybean nodulation. Frontiers in Rhizosphere Research. Columbia, MO (Abstract 33).
- Papers from 2005 to 2009
- Subramanian S, Cho U-H, Keyes CA, Yu O (2009) Proteomic analysis of soybean xylem sap in response to symbiotic and pathogenic interactions. BMC Plant Biology, submitted.
- Gutierrez-Gonzalez JJ, Guttikonda SK, Aldrich D, Tran PLS, Yu O, Nguyen HT, Sleper DA (2009) Differential expression of isoflavone biosynthetic genes in soybean during water stresses. BMC Genetics, submitted.
- Gutierrez-Gonzalez JJ, Wu X, Zhang J, Lee JD, Ellersieck M, Shannon GJ, Yu O, Nguyen HT, Sleper DA (2009) Genetic control of soybean seed isoflavone content: Importance of statistical model and epistasis in complex traits. Theoretical and Applied Genetics, accepted.
- Keyes CA, Subramanian S, Yu O (2008) Hairy root as a model system for undergraduate laboratory curriculum and research. Bioscene, accepted.
- Zhang J, Yu O (2008) Metabolic engineering of isoflavone biosynthesis in seeds. In Modification of seed composition to promote health and nutrition. Hari Krishnan (ed). Agronomy Monograph Series, pp 151-177.
- Zhang J, Subramanian S, Stacey G, Yu O (2008) Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti. The Plant Journal, 57: 171-183.
- Graham TL, Graham MY, Subramanian S, Yu O (2007) RNAi silencing of genes for elicitation or biosynthesis of 5-deoxyisoflavonoids suppresses race-specific resistance and HR cell death in Phytophthora sojae infected tissues. Plant Physiology 144: 724-740.
- Yamaguchi M, Valliyodan B, Zhang J, LeNoble ME, Yu O, Nguyen HT, Sharp RE (2007) Proteomic analysis of the differential response of elongation rate to water deficit within the soybean primary root growth zone. Plant Biology 2007, Chicago, IL. ASPB abstract.
- Halls C, Schroeder AC, Kumaran S, Cahoon RE, Jez JM, Yu O (2007) Metabolic engineering of the red wine compound resveratrol. The ASA-CSSA-SSSA International Annual Meetings, November, 2007, New Orleans, LA.
- Graham TL, Graham MY, Subramanian S, Yu O (2007) Use of Gene Silencing and Metabolomics to Characterize Interactive Stress and Defense Pathways in Soybean. 3rd Cell Stress Society International Congress on Stress Responses in Biology and Medicine. August 2007, Budapest, Hungary.
- Gutierrez J, Sleper D, Nguyen HT, Valliyodan B, Yu O (2006) Genetic Analysis of isoflavone accumulation during soybean seed development under drought stress. The ASA-CSSA-SSSA International Annual Meetings, November, 2006. Indianapolis, IN.
- Cheng H, Yu O, Yu D (2008) Polymorphisms of IFS1 and IFS2 genes are associated with isoflavone concentrations in soybean seeds. Plant Science, 175: 505-512.
- Graham TL, Graham MY, Yu O (2008) Genomics of secondary metabolism in soybean. In Genetics and Genomics of Soybean. Gary Stacey (ed.), Springer, New York. pp. 211-242.
- Yu O, Jez JM (2008) Nature's assembly line: Biosynthesis of simple phenylpropanoids and polyketides. Plant Journal 54: 750-762.
- Li L, He H, Zhang J, Wang X, Bai S, Stolc V,Tongprasit W, Young ND, Yu O, Deng XW (2008) Transcriptional analysis of highly syntenic regions between Medicago truncatula and Glycine max using tiling microarrays. Genome Biology, 9: R57. (http://genomebiology.com/2008/9/3/R57).
- Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O (2008) Novel and nodulation-regulated microRNAs in soybean roots. BMC Genomics 9: 160 (http://www.biomedcentral.com/1471-2164/9/160). Highly accessed article.
- Subramanian S, Stacey G, Yu O (2007) Distinct, critical roles of flavonoids during determinate and indeterminate legume nodulation. Trends in Plant Science 12: 282-285.
- Chabaud M, Boisson-Dernier A, Zhang J, Taylor CG, Yu O, Barker DG (2006) Agrobacterium rhizogenes-mediated root transformation. In The Medicago truncatula handbook. Ulrike Mathesius (ed), http://www.noble.org/MedicagoHandbook/.
- Subramanian S., Stacey G., Yu O (2006) Endogenous isoflavones are essential for soybean-Bradyrhizobium japonicum interactions. Plant Journal 48: 261-273.
- Schroeder AC, Kumaran S, Hicks LM, Cahoon RE, Halls C, Yu O, Jez JM (2008) Contributions of conserved serine and tyrosine residues to catalysis, ligand binding, and cofactor processing in the active site of tyrosine ammonia lyase. Phytochemistry 69: 1496-1506.
- Halls, C, Yu O (2008) Potential for metabolic engineering of resveratrol biosynthesis. Trends in Biotechnology, 26:77-81.
- Zhang J, Subramanian S, Zhang Y, Yu O (2007) Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation. Plant Physiology 144: 741-751
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Progress 11/15/06 to 11/14/07
Outputs
We continued the study of structures of multienzyme complexes in the flavonoid and isoflavonoid biosynthetic pathway. Previous reports suggested that many of the key enzymes in this pathway forms metabolic channels that direct the flow of substrates and intermediates between consecutive enzymes. Over the past 12 months, new data collected support our original hypothesis. Some of these data were reported in our five publications so far this year. Some of the unpublished ones were summarized in the four manuscripts currently under review, and will be briefly summarized here. By genetically manipulate isoflavone pathways in soybean to study the flow of pathway intermediates, we discovered many interesting and surprising functions of isoflavones in soybean. First, during the defense responses, isoflavones not only function as phytoalexins, but also play important roles in regulating programmed cell death during incompatible hypersensitive responses (Graham et al. 2007).
Second, during the sobean-Bradyrhizobium interaction, we demonstrated that isoflavones functions both inside and outside of the roots. Isoflavones may function as an internal Nod factor inducer that guided the invading rhizobia to produce Nod factors and initiate nodule primordial development (Subramanian et al. 2007). We continued this research this year by genetically engineer M. truncatula flavones and isoflavone biosynthesis. We tried to silence four different pathway enzymes. IFS silencing: Formononetin and biochanin A are the most abundant isoflavones in the roots Transgenic roots expressing the IFS RNAi construct, the total isoflavone levels in root extracts were reduced by 6 folds. We did not observe significant changes in the levels of other flavonoid compounds in IFSi roots suggesting that silencing of IFS did not affect the biosynthesis of other flavonoid compounds significantly. CHR silencing: In the CHR RNAi roots dihydroxyflavone were drastically reduced. Approximately
90% of the CHRi transgenic roots also had lower level of formononetin. Meanwhile naringenin, the precursor of biochanin A, increased in CHRi roots, suggesting that blocking the 5-deoxyflavonoid synthesis channeled chalcone substrates toward 5-hydroxylflavonoid accumulation. FNS silencing: In these roots, the levels of dihydroxyflavone were reduced significantly whereas the levels of isoflavones and other flavonoids were unaffected. Other major flavones in M. truncatula seeds, apigenin and luteolin were not detectable even in the wild type roots. CHS silencing: All flavonoids in the hairy roots were significantly reduced. Our results are comparable to a recent report where RNAi silencing of MtCHS was successful. The above data suggested close connections exist between flavonoid and isoflavonoid pathways. Silencing of CHR and FNS both increased 5-hydroxyflavonoid accumulations. These discoveries provide important foundations for our future enzyme interactions studies. We will be
interested in studying how the two types of CHI enzymes function in these two pathways. Many interesting findings related to nodulation and lateral root development were summarized in a manuscript submitted to PNAS lately.
Impacts
In addition to above mentioned works, we have also continued our study on the metabolic channeling in yeast resveratrol biosynthesis. We discovered that one of the limiting factors for de novo yeast resveratrol accumulation is the codon usage of key metabolic enzymes. The entry point enzyme TAL was isolated from Rhodobacter bacteria, therefore, having a poor codon usage in yeast. When we replaced two stretches of codons that have less than 10% representations of tRNAs, we were able to increase resveratrol accumulation significantly. When the entire coding region is re-synthesized with favorite codons, we detected de novo biosynthesis that accumulated to very high levels. These data suggested that gene expression, together with enzyme interactions, control secondary metabolite biosynthesis in heterologous systems. Taken together, our recent discoveries suggested that for successful metabolic engineering, multiple levels of regulation, including the enzyme activity,
pathway fluxes, and enzyme-enzyme interactions, need to be taken into considerations.
Publications
- Zhang J, Subramanian S, Stacey G, Yu O (2007) Different flavonoids have distinct functions during nodulation of Medicago truncatula by Sinorhizobium meliloti. Proceedings of the National Academy of Sciences, submitted.
- Yu O, Jez JM (2007) Nature's assembly line: Biosynthesis of simple phenylpropanoid and polyketide. Plant Journal, in press.
- Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O (2007) Novel and nodulation-regulated microRNAs in soybean roots. Genome Biology, submitted.
- Li L, He H, Zhang J, Wang X, Bai S, Stolc V,Tongprasit W, Young ND, Yu O, Deng XW (2007) Transcriptional analysis of highly syntenic regions between Medicago truncatula and Glycine max using tiling microarrays. Genome Biology, submitted.
- Graham TL, Graham MY, Yu O (2007) Genomics of secondary metabolism in soybean. In Genomics of soybean. Gary Stacey (ed), in press.
- Halls, C, Yu O (2007) Potential for metabolic engineering of resveratrol biosynthesis. Trends in Biotechnology, invited review, in press.
- Schroeder AC, Kumaran S, Hicks LM, Cahoon RE, Halls C, Yu O, Jez JM (2007) Contributions of conserved serine and tyrosine residues to catalysis, ligand binding, and cofactor processing in the active site of tyrosine ammonia lyase. Phytochemistry, in press.
- Zhang J, Subramanian S, Zhang Y, Yu O (2007) Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation. Plant Physiology 144: 741-751
- Graham TL, Graham MY, Subramanian S, Yu O (2007) RNAi silencing of genes for elicitation or biosynthesis of 5-deoxyisoflavonoids suppresses race-specific resistance and HR cell death in Phytophthora sojae infected tissues. Plant Physiology 144: 724-740.
- Subramanian S, Stacey G, Yu O (2007) Distinct, critical roles of flavonoids during determinate and indeterminate legume nodulation. Trends in Plant Science 12: 282-285.
- Chabaud M, Boisson-Dernier A, Zhang J, Taylor CG, Yu O, Barker DG (2006) Agrobacterium rhizogenes-mediated root transformation. In The Medicago truncatula handbook. Ulrike Mathesius (ed), http://www.noble.org/MedicagoHandbook/.
- Subramanian S., Stacey G., Yu O (2006) Endogenous isoflavones are essential for soybean-Bradyrhizobium japonicum interactions. Plant Journal 48: 261-273.
- Zhang Y, Liu ZH, Jia L, Peng ZL, Jaworski J, Wang XM, Jez J., Chen F, Yu O (2006) Metabolic engineering of de novo biosynthesis of resveratrol in Saccharomyces cerevisiae and mammalian cells. Journal of American Chemistry Society. 128: 13030-13031. Featured in Chemical and Engineering News, 84 (Oct. 2, 2006): page 43.
- Ralston L, Yu O. (2006) Metabolons involving plant cytochrome P450s. Phytochemistry Review. 5: 459-472.
- Yu O. (2006) Metabolic engineering of the plant phenylpropanoid pathway. Encyclopedia of Plant and Crop Science, Dekker Publishing. DOI: 10.1081/E-EPCS-120010589.
- Yu O, Matsuno M, Subramanian S. (2006) Flavonoids in flowers: Genetics and Biochemistry. In Floriculture, ornamental and plant biotechnology: Advances and topical issues (1st Edition), Jaime A Teixeira da Silva (ed.), pp. 283-293.
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Progress 11/15/05 to 11/15/06
Outputs
The focus of the project is to understand the organization of key metabolic enzymes involved in the flavonoid and isoflavonoid biosynthesis. Preliminary evidence showed several enzymes form a metabolic channel (or "metabolon") on the cytoplasmic surface of ER. This enzyme complex is anchored by cytochrome P450 enzymes, and functions to channel intermediate substrates from one enzyme to the next, reducing their release to the cytosols. We have established a yeast system by reconstituting the plant phenylpropanoid pathway. We introduced chalcone isomerase (CHI), isoflavone synthase (IFS), flavanones 3-hydroxylase (F3H), and flavone synthase (FNS) into the yeast, separately or in combination. This functional assay system allows us to investigate enzyme interactions in a defined environment. During this funding period, we have repeated several key experiments that demonstrated direct protein-protein interactions in yeast. We have again shown that type II CHI can interact
with IFS by co-immunoprecipitation assay and tandem affinity purifications. However, we still have difficulty to quantify the observed FRET signals in FLIM analysis. We are currently focusing on the microscopy part of the project and trying to collect solid data for the main publication. We have studied the other partner of CHI, FNS in more details. FNS competes with IFS for naringenin. We cloned two FNS genes from Medicago truncatula, and carried out detailed functional analysis of the enzyme. We found that Medicago FNS is 2-hydroxyflavanone hydroxylase. Unlike non-legume FNS, it did not complete the dehydration reaction to form flavone. We have tried to identify the dehydratase of the 2-hydroxyflavone by expressing two dehydratase homologs. One of FNS gene is highly induced by defense signals. We show that RNAi silencing of Medicago FNS significantly reduces its potential for nodulation. The manuscript reporting these discoveries has been accepted by Plant Physiology. Resveratrol is
a naturally occurring defense compound produced by a limited number of plants. This health-promoting compound can extend life spans in yeasts, flies, worms, and fishes. Resveratrol biosynthesis is part of the flavonoid pathway. To biosynthesize resveratrol de novo, tyrosine ammonia lyase (TAL), p-coumarate CoA-ligase (4CL), and stilbene synthase (STS) were isolated from Rhodobacter sphaeroides, Arabidopsis thaliana, and Vitis vinifera, respectively. Yeast cells expressing 4CL and STS can produce resveratrol in large quantities when fed with p-coumaric acid. Because enzyme interactions increase the flux of the pathway towards final products, when a translational fusion protein joining 4CL and STS was used, yeast cells produced up to 15 folds higher resveratrol than the co-transformed cells, suggesting that physical interactions of 4CL and STS facilitate resveratrol production. When the resveratrol pathway was introduced into a mammalian host, the human HEK293 cells, de novo
biosynthesis was detected, leading to intracellular accumulation of resveratrol. We successfully engineered an entire plant natural product pathway into a mammalian host. This work has been published by JACS.
Impacts
Understanding the organization of complex metabolic pathway is very important to understand and engineer biosynthesis process. As shown in one of publications, joining separate enzymes together can increase the production of final product significantly. Our work is highlighted in an issue of Chemical and Engineering News (C&E News 2006, Oct 2, page 43). Our work in resveratrol has generated many interested in different companies. We are currently in discussion with two California based company and one Missouri based company to license our technology and to solicitate their support in my research. We have also submitted a proposal to CPBR seeking jointed Federal and commercial funding opportunities.
Publications
- Graham TL, Graham MY, Yu O (2007) Genomics of secondary metabolism in soybean. In Genomics of soybean. Gary Stacey (ed), submitted.
- Zhang J, Subramanian S, Zhang Y, Yu O (2007) Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation. Plant Physiology, accepted.
- Subramanian S, Stacey G, Yu O (2007) Distinct, critical roles of flavonoids during determinate and indeterminate legume nodulation. Trends in Plant Science, submitted.
- Chabaud M, Boisson-Dernier A, Zhang J, Taylor CG, Yu O, Barker DG (2006) Agrobacterium rhizogenes-mediated root transformation. In The Medicago truncatula handbook. Ulrike Mathesius (ed), submitted.
- Subramanian S, Stacey G, Yu O (2006) Endogenous isoflavones are essential for soybean-Bradyrhizobium japonicum interactions. Plant Journal 48: 261-273.
- Zhang Y, Liu ZH, Jia L, Peng ZL, Jaworski J, Wang XM, Jez J., Chen F, Yu O (2006) Metabolic engineering of de novo biosynthesis of resveratrol in Saccharomyces cerevisiae and mammalian cells. Journal of American Chemistry Society. 128: 13030-13031.
- Ralston L, Yu O. (2006) Metabolons involving plant cytochrome P450s. Phytochemistry Review 5: 459-472.
- Yu O. (2006) Metabolic engineering of the plant phenylpropanoid pathway. Encyclopedia of Plant and Crop Science, Dekker Publishing. DOI: 10.1081/E-EPCS-120010589.
- Yu O, Matsuno M, Subramanian S. (2006) Flavonoids in flowers: Genetics and Biochemistry. In Floriculture, ornamental and plant biotechnology: Advances and topical issues (1st Edition), Jaime A Teixeira da Silva (ed.), pp. 283-293.
- Graham TL, Graham MY, Subramanian S, Yu O (2007) RNAi silencing of genes for elicitation or biosynthesis of 5-deoxyisoflavonoids suppresses race-specific resistance and HR cell death in Phytophthora sojae infected tissues. Plant Physiology, submitted.
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