Executive Summary:

Isoflavones are a group of phenolic compounds found mostly in legumes. They have molecular structures similar to human hormone estrogen. Many documented health benefits associated with consumption of legumes (particularly soybeans) have been linked to isoflavones. Engineering non-legume crops such as maize to produce isoflavones will significantly enhance their nutritional values. During the project period, 1) we have successfully produced transgenic maize carrying soybean isoflavonoid enzymes and producing isoflavones in the kernels. We found that the isoflavone biosynthetic enzymes depend on endogenous phenylpropanoid pathway for their substrate, naringenin; at the same time, it must compete with other downstream flavonoid enzymes for the same substrate to produce high levels of isoflavones. 2) We also investigated the bottlenecks of isoflavonoid metabolic engineering. We studied specific enzyme interactions and enzyme localizations. We found that multiple enzymes of a pathway could form metabolic channels. These channel complexes control the access of substrates and influence the flux of intermediates. In addition, flavonoid and isoflavonoid enzymes appear to form their own metabolic channels separately. We were able to reconstitute the entire biosynthetic pathway in yeast, and utilized fusion proteins that joint different enzymes into one protein to increase product yields under in vivo conditions. The results related to this work were published in six peer-reviewed papers and supported us to successfully obtain federal fundings in four grant proposals.

Research Activities:

1. Characterization of IFS transformed maize.

In legumes, isoflavone synthase (IFS) is the entry point enzyme that directs flavonoid intermediates, such as naringenin, to isoflavones by a unique aryl-migration reaction. We have transformed maize with an IFS gene driven by seed specific oleosin promoter. To increase the flux of intermediates in the phenylpropanoid pathway, this transgenic maize also carries a Myb-like transcription factor CRC. This transcription factor up-regulates most of the phenylpropanoid pathway genes, thus providing the IFS-substrate naringenin in a tissue-specific manner. However, CRC also enhances the expression of many downstream enzymes that convert naringenin to various flavonoid compounds, as evidenced by the intense anthocyanin accumulation in the embryos. As a result, the accumulation of isoflavones is significantly lower in transgenic maize than in soybean tissues.

To overcome this problem, the maize a1 mutant that is defective in a flavonoid pathway enzyme DFR was crossed to IFS and CRC co-transformed transgenic maize. DFR is responsible for initiating 3-deoxyflavonoid phytoalexin biosynthesis in maize and is involved in anthocyanin production as well. We introduced this mutation to block the competing flavonoid pathway and to re-direct the flux of substrates towards isoflavone production. The individual F3 (or T3) seedlings showed segregation of red-leaf phenotype because the transgenic plants carrying a1 mutant did not accumulate red pigments in the leaves or stems. The phenolic compounds from these plants were extracted and analyzed by HPLC. However, no significant changes in the total isoflavone level were detected; suggesting DFR alone did not block the flavonoid pathway entirely. The HPLC analysis conducted on F4 (or T4) seeds showed similar results.

One of the hypotheses for this observation is that unlike in Arabidopsis, there are at least four other enzymes in maize that use naringenin as a substrate, including flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3', 5'-hydroxylase (F3'5'H), and flavone synthase (FNS-II). Blocking DFR may have re-directed the flow of substrate towards other flavonoid pathways, as shown by the altering HPLC profile. It also suggests that the heterologously expressed IFS might have difficulty to compete efficiently with endogenous enzymes. These information were discussed in our Phytochemistry paper, and in the paper published in Advances in Agronomy.

2. The bottlenecks of isoflavone engineering.

Based on above observations, we believe that to achieve high level of isoflavone accumulations in maize, we need to investigate the factors that limits the flow of intermediates towards isoflavone biosynthesis. We focused on the junction of these two competing pathways. Naringenin is shared by both flavonoid and isoflavonoid pathway. The enzyme that synthesizes naringenin is chalcone isomerase (CHI). Two types of CHIs exist in higher plants. Type I CHIs are broadly distributed in higher plants and convert naringenin-chalcone into naringenin. Type II CHIs are legume-specific and in addition to catalyzing the previous reaction, convert isoliquiritigenin to liquiritigenin, which serves as the substrate for the biosynthesis of isoflavone daidzein. Previously, we introduced chalcone reductase (CHR) into maize to produce isoliquiritigenin, and eventually daidzein. Together with IFS and CRC, the transgenic maize produced only minute amounts of daidzein detectable only by GC-MS. 

One of the limiting factors is likely to be the lack of type II CHI in maize genome.  The isoliquiritigenin produced by CHR may not be efficiently converted to liquiritigenin. We identified seven EST sequences from soybean EST database that are homologous to CHI. Gene expression and kinetics analysis suggest that the type I CHI is coordinately regulated with other flavonoid-specific genes. While the type II CHIs are coordinately regulated with isoflavonoid-specific genes. The identification and characterization of type II CHI in soybean may eventually allow high level of engineered daidzein production in maize. These data were reported in our Plant Molecular Biology paper.

Another hypothesis to explain the low isoflavone accumulation in transgenic maize is that the IFS may not interact with endogenous CHI efficiently (Type I). We tested this hypothesis by studying the enzyme interactions between IFS and CHI. First we reconstituted the isoflavonoid pathway in yeast. When one of the type II CHIs was co-expressed with IFS, various chalcone substrates added to the culture media were efficiently converted to an assortment of isoflavanones and isoflavones. Then we reconstructed the flavonoid pathway by co-expressing type I CHI with either F3H or FNS II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux. These data were reported in our Plant Physiology paper.

When IFS was fused with an YFP florescent protein at the C-terminus and the type II CHI was fused with an CFP at the N-terminus, the two fusion proteins could be co-transformed into yeast. If the IFS-CHI interaction exists, a FRET signal that arose from CFP excitation of the YFP would be detected. As expected, we not only detected the YFP signal by CFP excitation, but also the enhanced CFP signal when the YFP was bleached out. This analysis provided strong evidence that two enzymes form close associations in yeast. We also confirmed these data by extensive immuno-precipitation assays and affinity purification assays. These results was presented at a mini-symposium in 2004 ASPB annual meeting.

In conclusion, we have attempted the stated goals of isoflavone production in maize. Various pathway engineering approaches were taken, including heterologous expression of soybean enzymes in maize, introducing transcription factors, crossing to maize mutant to block the competing pathways. Isoflavones were detected in maize kernels. More importantly, we investigated the effects of metabolic channeling on the flux of intermediates. We built a yeast in vivo system to study enzyme interactions. Understanding the mechanisms and bottlenecks of isoflavonoid engineering will lead to novel approaches for future improvement of maize nutritional values.

Publications and Presentations during the Project Period:

Papers:

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.

Subramainan 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.

Bennett JO, Yu O, Heartherly LG, Krishnan HB. (2004). Accumulation of genistein and daidzein, soybean isoflavones implicated in promoting human health, is significantly elevated by irrigation. Journal of Agricultural and Food Chemistry 52: 7574-7579.

Subramanian S, Xu L, Lu G, Odell J, Yu O. (2004) The promoters of the isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots. Plant Molecular Biology 54:226-239.

Yu O, Shi J, Hession A, Maxwell C, McGonigle B, Odell J. (2003) Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochemistry 63: 753-763

Presentations:

Matsuno M, Ralston L, Subramanian S, Walker L, and Yu O. 2004. In vivo reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using type I and type II chalcone isomerases from soybean. Invited for oral presentation at the 2004 ASPB Annual Meeting minisymposium-Secondary Metabolism.

Subramanian S, Menne C, Odell JT, Stacey G, and Yu O. 2004. Isoflavones are essential for the establishment of symbiosis between soybean and Bradyrhizobium japonicum. Invited for oral presentation at the 2004 ASPB Annual Meeting minisymposium-Symbiosis.

Subramanian S, Matsuno M, Menne C, Ralston L, and Yu O. 2004. From transcriptional regulation to metabolic channeling: Understanding the flavonoid and isoflavonoid pathway for metabolic engineering. Invited for oral presentation at the Soy 2004 Meeting at Columbia, MO.

Matsuno M, Ralston L, Subramanian S, Walker L, and Yu O. 2004. Metabolic engineering of phytoestrogenic compounds - isoflavones. Invited for oral presentation at the International Symposium on "Profiling and the use of plant metabolites and medicinal phytocompounds" at Taipei, Taiwan.

Patent:

Ralston L and Yu O. 2003. In vivo reconstruction of the flavonoid and isoflavonoid pathway. Technology Disclosure from the Donald Danforth Plant Science Center.

Invited Talks:

Metabolic engineering of soybean phytoestrogens. Presented at the International Symposium on Research into Plant Metabolites and Medicinal Phytocompounds. Taipei, Taiwan. Dec 2004.

Metabolic regulations of isoflavonoid biosynthsis. Dept. of Biochemistry, Fudan University, Shanghai, China. Dec. 2004

The biological functions of isoflavones during soybean Bradyrhizobium interactions. Inst. Of Botany, Academia Sinica. Dec. 2004.From transcriptional regulation to metabolic channeling: metabolic engineering of flavonoid and isoflavonoid biosynthesis. Presented at the Soy 2004 Symposium. Columbia, MO. Aug 2004.

In vitro reconstruction of flavonoid and isoflavonoid pathway in yeast. Presented at the ASPB Annual Meeting. Orlando, FL. July 2004.

Isoflavones are essential during soybean bradyrhizobium interactions. Presented by Senthil Subramanian (postdoc) at ASPB Annual Meeting. Orlando, FL, July 2004.

Other Information for IMBA Record:

Oliver Yu was interviewed by St. Louis local NBC TV station (KSDK Channel 5) and appeared in the evening News program on 10/16/2002, in a segment titled "Health Benefits of Soy", to introduce the research projects conducted in his lab. 

Oliver Yu was the organizer and host of "Soybean Phytoestrogen Research Meeting" at the Danforth Center, May 2005. Fourteen speakers from eight universities were invited to discuss progress and challenges in soybean isoflavone research, and to explore funding and collaboration opportunities. We obtained financial support from the United Soy Board for this meeting.

Additional Federal Funding Obtained During the Project Period:

Illinois Missouri Biotechnology Alliance (USDA-IMBA). Oct. 1, 2004 - Apr. 30, 2006. "Identification of Key Defense Genes for Resistance to Phytophthora Stem and Root Rot Through Gene Silencing". PI: Terry Graham, co-PI: Lian-mei Graham, Oliver Yu. $79,540 (out of $170,000 total).

NSF Major Research Instrumentation. July 24, 2004. "Acquisition of a laser capture microdissection system". PI: Chris Taylor, co-PI: Howard Berg, Mark Running, Oliver Yu, and Ralph Quatrano. $165,894.

NSF Major Research Instrumentation. July, 2005. "Acquisition of LC-MS for Plant Metabolic Profiling". PI: Xuemin Wang, co-PI: Liming Xiong, Oliver Yu, Daniel Schachtman, Jan Jaworski. $500,090.

USDA 1890 Institution Capacity Grant. Nov. 2005 - Oct, 2008. "Metabolic Engineering of Isoflavone in Rice". PI: Muthusamy Manoharan (Univ. of Arkansas at Pine Bluff), co-PI: Oliver Yu, Yulin Jia, James Garnerr. $24,000 (out of $210,000).

NSF Metabolic Engineering Working Group. Nov, 2005 - Oct, 2008. "Organization of Isoflavonoid Biosynthetic Enzymes". $450,000 (NSF Metabolic Biochemistry $200,000; USDA Biochemistry $250,000).