Progress 06/01/20 to 05/09/25
Outputs
Target Audience:The target audience of this plant synthetic biology project is broad, from agriculture industry or food and beverage industry that are interested in improving crop nutritional values (e.g. betalain pigments and vitamin E contents) as well as pharmaceutical industry that would like to improve the production of plant natural products (e.g. morphine and codeine, isoquinoline alkaloids derived from tyrosine) without compromising the overall crop yield. Toward this target audience in mind, we started experiments in model plants, such as Arabidopsis and Nicotiana, but then we also carried out studies in crops, such as tobacco, and soybean to test transformability of our metabolic engineering strategy. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for scientist, graduate and undergraduate students to be trained in the interdisciplinary field of basic plant biochemistry and synthetic biology. Amy Yi (previous high-school intern; continued working as an undergraduate student) and Elizabeth Siaw (undergraduate student) have been working to validate the role of candidate genes of the vacuolar transporter. Soyoung Jung (graduate student) and Marcos de Oliveira (scientist) have been working for optimizing betalain constructs and generating stable transgenic lines in different plant species. We met weekly to discuss progress and next experimental plans. They presented their findings at the lab meeting, on-campus seminar series, and national/international conferences. Through the preparation of one research publication and one patent, Soyoung Jung learned how to present scientific work to both academic and more broader industrial communities. The PI Maeda also discussed with Soyoung and Marcos about their career goals at the beginning of the year using their individual developmental plan (IDP). How have the results been disseminated to communities of interest?Our first finding has been already published in a peer-reviewed journal (Jung and Maeda, 2024). Additionally, we are preparing at least two more manuscripts to be submitted soon (one review and one research papers). Soyoung Jung attended the following three meetings to share our recent findings with the science community: Debottlenecking the L-DOPA 4,5-dioxygenase step with enhanced tyrosine supply boosts betalain production in Nicotiana benthamiana. Plant Biology 2024, Honolulu, HI, June 22-26, 2024 poster presentation Debottlenecking the L-DOPA 4,5-dioxygenase step with enhanced tyrosine supply boosts betalain production in Nicotiana benthamiana. Midwest ASPB 2024 Meeting from March 16th to 17th at Purdue University poster presentation We also carried out in-person outreach events to disseminate our work to the general public and the importance of plant pigments and other metabolites to human well-being and society. At the UW Science Expedition on campus and Science Night events at various elementary schools in the city of Madison from 2020 to 2025, we carried out in-person outreach event, where children carried out chromatography using different plant pigments, including betalain pigments, to learn about their properties and their importance in our society. We also generated a "metabolic pathway" jigsaw puzzle for children to learn how different pigments are made from CO2 and sunlight energy through plant metabolic pathways. We also attended USDA Agrifood Innovation Symposium 2024 to interact with agricultural and food system innovators, scientific experts, and thought leaders and showcase the cutting-edge discoveries made possible by both longstanding and new investments in public agricultural research and development. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
IMPACT: Betalains--natural red pigments predominantly found in beets--are widely used in the food industry. However, current production methods relying on beet extraction suffer from low yield and high costs. As consumer demand shifts toward clean-label products and regulatory bans on synthetic food dyes (e.g., Red 3 and Red 40) intensify, there is an urgent need for a sustainable, scalable alternative for natural red pigment production. Our work directly aligns with industry priorities to increase pigment concentration and reduce production costs. Additionally, it advances the broader field of plant synthetic biology, where challenges such as pathway optimization, precursor availability, and chassis selection remain significant barriers. Over the past five years, we have addressed these challenges by identifying key bottlenecks and demonstrating how precursor supply and host-specific metabolism impact betalain production efficiency in planta. Notably, our high-betalain soybean lines also accumulated elevated levels of other aromatic compounds, including L-DOPA, a compound with pharmaceutical applications (e.g., Alzheimer's treatment). This research establishes a robust framework for scalable, sustainable production of valuable plant-derived compounds. Key Outcome: We first investigated how enhancing tyrosine supply affects the production of tyrosine-derived compounds like betalains in plants. Previous studies showed that low tyrosine levels limit the production of betalains in Arabidopsis. However, since tyrosine is essential for plant development, its overaccumulation can disrupt growth. Overexpression of a partially-deregulated tyrosine-synthesizing TyrA enzyme from Beta vulgaris (BvTyrAα) elevated tyrosine levels but led to growth defects in Arabidopsis. To balance tyrosine supply without compromising plant growth, we used a "push-pull strategy". This involved overexpressing BvTyrAα ("push") to boost tyrosine levels while introducing betalain biosynthetic genes ("pull") to direct tyrosine toward betalain production. To test this strategy, we generated recombinant DNA constructs overexpressing i) BvTyrAα ("push"), ii) three betalain biosynthetic genes ("pull"), iii) BvTyrAα and betalalin genes ("push/pull"), and iv) empty vector (EV) and transiently expressed those constructs in Nicotiana benthamiana leaves using agroinfiltration. Unexpectedly, leaves expressing the initial "push/pull" construct exhibited lower pigmentation than those expressing the "pull" only construct. Metabolite analysis revealed that while tyrosine and L-DOPA (a betalain pathway intermediate) levels were significantly increased in the "push/pull" lines, this did not translate to higher betalain levels, indicating the presence of a bottleneck within the "pull" (betalain) pathway. The expression of the "push/pull" construct also failed to produce stable transgenic tobacco and soybean lines, likely due to accumulation of a toxic intermediate resulting from the bottleneck. To identify a rate limiting step(s) of betalain biosynthetic pathway, we co-expressed individual betalain biosynthetic gene (i.e., CYP76AD1, DODA, or cDOPA5GT) with the previously generated constructs (i.e., "push", "pull", "push/pull", or EV). Among these, co-expression of DODA (L-DOPA 4,5-dioxygenase) with betalain constructs produced significantly higher betalain levels, identifying DODA as a critical bottleneck. Interestingly, only when additional DODA construct was co-expressed, higher pigmentation was observed in the leaves expressing the "push/pull" construct than the "pull" construct, suggesting that enhancing precursor supply ("push") can lead to increased target compound production when the target downstream pathway ("pull") is optimized (Jung and Maeda, 2024, Plant Physiol.). Next, to overcome this limitation, we optimized the "pull" construct by modifying the previously published RUBY construct, where three betalain genes were expressed polycistronically. As the first gene that positions next to the promoter shows the highest expression level in the polycistronic system, we rearranged the order of the coding sequences to have the DODA gene in the first position in our new RUBY version 2 (RUBYv2) construct. We also added an additional DODA transcriptional unit to RUBYv2 to further debottleneck the DODA step. We also adjusted the "push" construct by using either a strong or weak promoter for BvTyrAα expression, generating "strong push+pull" and "weak push+pull" constructs. To test the impact of betalain production in various plant hosts, we generated stable transgenic lines using Arabidopsis, tobacco and soybean with the newly generated constructs. In all three species, "push+pull" lines produced higher betalain levels than "pull" lines. However, Arabidopsis and tobacco "strong push+pull" lines showed growth defects, likely due to high accumulation of tyrosine and an intermediate, L-DOPA. In contrast, soybean "push+pull" lines maintained normal growth despite enhanced tyrosine and L-DOPA levels, suggesting greater tolerance of soybean to these compounds. Metabolite analyses using the mature leaves showed accumulation of betalains up to 8.53 mg/gDW (1.28 mg/gFW) in "push+pull" soybean lines, which is significantly higher than betalain production in beets (Jung et al., in preparation, Jung et al., 2025 US provisional patent filing). Redirecting available precursor to certain subcellular localization can boost target compound production in plant synthetic biology (Jung et al., review article in preparation). Our previous studies revealed that most of the tyrosine in BvTyrAα-overexpressing Arabidopsis lines was sequestered in vacuoles (Maeda et al., in preparation), limiting its availability for tyrosine-derived compound production. To better understand this interface between primary and specialized metabolism, we also performed RNA-seq analysis using two BvTyrAα-overexpressing lines (i.e., #10 and #23) in comparison to an empty vector (EV) control. We found two putative vacuolar tyrosine transporters, organic cation/carnitine transporter5 (AtOCT5, AT1G79410) and multidrug resistance-associated protein 13 (AtMRP13, AT1G30410), upregulated in both BvTyrAα overexpressing lines compared to the EV. To investigate their role, we have isolated Arabidopsis T-DNA knockout mutant lines of AtOCT5 and AtMRP13 and crossed them with the BvTyrAα overexpression lines. Metabolite analysis showed the increased levels of tyramine, a tyrosine-derived compound, in a crossed line with mrp13 and BvTyrAα-overexpressing line. While further characterization of these crossed lines are needed, reducing the vacuolar sequestration of the tyrosine precursor may enhance production of tyrosine-derived compounds, including betalain pigments. Implication of the results: Our findings directly addressed the two main questions of this project. First, our date demonstrate that primary metabolite precursor supply substantially contribute to the efficient production of natural products, though only after the downstream pathway is optimized. By identifying and optimizing the DODA-catalyzed step as a critical bottleneck in the betalain biosynthetic pathway, enhancing tyrosine supply resulted in a dramatic increase in betalain production in planta. Second, to determine the most suitable plant platforms for high-level betalain production, we generated transgenic lines in soybean, tobacco, and Arabidopsis. Among these, soybean demonstrated high betalain accumulation without compromising its overall growth, exceeding levels found in beets without exhibiting growth defects, highlighting its suitability for sustainable betalain production. This work advances our ability to design efficient and scalable plant-based systems for sustainable production of valuable plant natural products through synthetic biology.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Plant Biology 2024, Honolulu, HI, June 22-26 (poster presentation) Title: Debottlenecking the L-DOPA 4,5-dioxygenase step with enhanced tyrosine supply boosts betalain production in Nicotiana benthamiana
Authors: Soyoung Jung and Hiroshi A. Maeda
|
Progress 06/01/23 to 05/31/24
Outputs
Target Audience:The target audience of this plant synthetic biology project is broad, from agriculture industry that are interested in improving crop nutritional values (e.g. betalain pigments and vitamin E contents) as well as pharmaceutical industry that would like to improve the production of plant natural products (e.g. morphine and codeine, isoquinoline alkaloids derived from tyrosine) without compromising the overall crop yield. Toward this target audience in mind, we started experiments in model plants, such as Arabidopsis and Nicotiana, but then we are also carrying out studies in crops, such as tobacco, soybean, and sorghum, to test transformability of our metabolic engineering strategy. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for a graduate student, Soyoung Jung, together with a scientist, Marcos de Oliveira, to be trained in the interdisciplinary field of basic plant biochemistry and synthetic biology. We met weekly to discuss progress and next experimental plans. Soyoung and Marcos have also presented their results at the lab meeting. The PI Maeda also discussed with Soyoung and Marcos about their career goals at the beginning of the year using their individual developmental plan (IDP). How have the results been disseminated to communities of interest?Soyoung Jung attended the following three meetingsto share our recent findings with the science community: Midwest ASPB 2024 Meeting from March 16th to 17th at Purdue University to share our recent findings with the science community. 2023 Plant Metabolic Engineering Gordon Research Seminar, June 10-11 2023 to share our recent findings with the science community. 2023 Plant Metabolic Engineering Gordon Research Conference, June 11-16 2023 to share our recent findings with the science community. We also carried out in-person outreach events to disseminate our work to the general public and the importance of plant pigments and other metabolites to human well-being and society. At the UW Science Expedition, we carried out in-person outreach event called "The world of plants", together with four other laboratories of the PlantCMB group on UW campus to maximize our visibility. Among ~50 booths in the Wisconsin Institute for Discovery, our plant outreach was one of the most popular booths with more than 170 people attended due to hands-on activities where children can actually try themselves to learn. In the Maeda lab table, children carried out chromatography using different plant pigments, including betalain pigments, to learn about their properties and their importance in our society. This year, we also generated a "metabolic pathway" jigsaw puzzle for children to learn how different pigments are made from CO2 and sunlight energy through plant metabolic pathways. What do you plan to do during the next reporting period to accomplish the goals?Our data from this past year showed that the "optimized pull" construct containing RUBYv2 with an additional DODA transcription unit can produce higher betalain content than the previous "pull" construct by alleviating the DODA bottleneck step. This coming year, first we are planning to better understand the impact of optimizing a target pathway in stable Arabidopsis transgenic line using the "optimized pull" construct. We already confirmed the pigmentation in T1 transgenic lines in both RUBYv2 and "optimized pull" lines. After selecting homozygous Arabidopsis T2 lines expressing either the "optimized pull" or RUBYv2 construct, we will further analyze metabolic and transcriptional responses using LC-MS and qPCR, respectively to see the impact of an additional DODA transcriptional unit on in planta betalain production. Only a few previous studies have generated stable transgenic lines to produce target compounds, and many of them had detected defects on growth or development in those stable transgenic likely due to the accumulation of toxic precursor or intermediates or metabolic trade-off. To better understand how balancing between supply and efficient utilization of precursor impacts on producing target compounds while not compromising their growth and development, we will further analyze the crossed lines generated using two BvTyrAα-overexpressing lines or either the RUBYv2 or "optimized pull"-expressing lines. We will first examine the phenotype of each crossed lines to see if additional precursor can boost betalain production or if any of them show any defects on their growth or development. Also, we will perform metabolite and transcriptional analysis to test the correlation between phenotype with metabolite and/or transcript levels. Finally, we will investigate the impact of using different plant chassis on production of a target compound. In microbial studies, it has been shown that choosing a suitable host species can dramatically increase the yield of final product. However, most plant synthetic biology works have been conducted using transient expression in N. benthamiana and, if any, only a few other plant species. To investigate the impact of choosing a suitable plant chassis, we will generate stable transgenic lines using Arabidopsis, tobacco and soybean with the above newly generated constructs. After successfully generating stable transgenic lines, we will compare different metabolic or transcriptional responses in various plant species to test which species is best suited for betalain production.
Impacts
What was accomplished under these goals?
Plants provide promising platforms to efficiently produce diverse chemicals sustainably. Yet, our knowledge of implementing plant synthetic biology is highly limited. In our previous reports, we identified that once the target biosynthetic pathway is optimized by alleviating rate limiting step(s), enhanced precursor supply boosts the target compound production. Based on these results, this year we generated optimized betalain construct, containing the RUBYv2 with additional DODA transcriptional unit, and tested the impact of tyrosine supply in two different plants, Arabidopsis thaliana and Nicotiana benthamiana. Furthermore, by varying the strength of the promoter to drive BvTyrAα expression, we have further tested the optimal level of tyrosine for improved betalain production in planta. Our findings represent a significant step forward in plant synthetic biology and have practical implications for producing high-value compounds in plants for use in various industries. Specific Accomplishments for each objective: Objective 1) Contribution of tyrosine supply to betalain production using Arabidopsis. Last year, we identified that the L-DOPA 4,5-dioxegenase (DODA) is the rate limiting step of the betalain biosynthetic pathway and achieved betacyanin yield of 2.87 mg/gFW by alleviating the DODA bottleneck step together with enhanced tyrosine supply, which was almost twice higher than that in the red beet. Our data suggested that once the target pathway is optimized, additional precursor supply boosts the in planta target compound production (Jung and Maeda, 2024, Plant Physiol.). Based on the finding, this year we first generated the "optimized pull" construct. The RUBY construct expresses three betalain biosynthetic genes polycistronically. Many of the previous studies have shown that the first gene that positions next to the promoter shows the highest expression level in the polycistronic gene expression system. Therefore, we rearranged the order of the coding sequences of three betalain biosynthetic genes and named it the RUBY version 2 (RUBYv2), having the DODA coding sequence right after the Arabidopsis Rubisco promoter (PAtRbcS3) followed by the cDOPA5GT and CYP76AD1 coding sequences. We also added an additional DODA transcriptional unit driven under the tomato Rubisco promoter (PSlRbcS2) as well as the RUBYv2 construct since our recently published data has shown that DODA step is also limited in the RUBY construct. This construct having RUBYv2 and an additional DODA transcriptional unit, driven by PAtRbcS3 and PSlRbcS2, respectively, was named as the "optimized pull" and used for further study. To test the impact of the additional DODA transcriptional unit on the betalain production, we generated transgenic Arabidopsis lines using either the RUBYv2, the "optimized pull", or empty vector (EV) control. As expected, Arabidopsis T1 plants expressing either the RUBYv2 or the "optimized pull" showed betalain pigments, but not in the EV. The intensity of pigments varied in different T1 individuals within the lines expressing the same construct, from the ones showing slight pigmentation only in their hypocotyls to the ones showing strong pigmentations in all tissues. We speculated that this various pigmentation could be due to different expression levels of transgenes because of the positional effect. To directly compare the impact of additional tyrosine supply on betalain production, we decided to cross either the RUBYv2 or "optimized pull"-expressing T1 individuals with BvTyrAα-overexpressing lines. To vary the level of tyrosine, we used two BvTyrAα-overexpressing lines (i.e., #10 and #23), accumulating around 150- and 300-fold more tyrosine respectively than the EV (de Oliveria et al., 2019, Plant J.). Thus far, we have successfully generated crossed lines using two BvTyrAα-overexpressing lines either with the RUBYv2 or "optimized pull"-expressing lines and will further analyze those crossed lines. Objective 2) Cross-species transformability of the betalains pathway reconstruction in various plants. In the past year, we investigated the impact of previous "push", "pull", or "push/pull" construct on stable tobacco (Nicotiana tabacum) or soybean (Glycine max) transgenic lines. In betalain pathway-expressing T0 "pull" tobacco and soybean plants, betalain pigmentation was detected, whereas no pigmentation was observed in EV lines, as expected. However, we could not obtain any "push/pull" T0 tobacco or soybean stable transgenic lines. Based on our recent findings in N. benthamiana leaves (Jung and Maeda, 2024, Plant Physiol.), this problem is likely due to the hyperaccumulation of a toxic intermediate, in particular, L-DOPA. To overcome this L-DOPA toxicity issue, we decided to use the newly generated "optimized pull" construct to better "pull" tyrosine and L-DOPA for producing betalains. Furthermore, to prevent the marginal expression of transgenes in prokaryotes, we inserted intron sequence in the BvTyrAα coding sequence to generate the new "push" construct. As coordinated transgene expression in a cell-type specific manner is important to efficiently produce target compounds in heterologous plant hosts, we used Arabidopsis light harvesting complex promoter (PAtLHB1B1) to express BvTyrAα (hereafter, named PAtLHB1B1-"push") at photosynthetic tissues, where PSlRbcS2 and PAtRbcS3 will express the additional DODA transcriptional unit and the RUBYv2, respectively in the "optimized pull" construct. Since PAtLHB1B1 is one of the "strong" constitutive promoters, we also generated another "push" construct using a promoter from the NADH dehydrogenase ubiquinone 1 beta subcomplex unit (AT1G76200, PCL1), previously identified as a constitutive promoter with low expression level (Zhou et al., 2023, ACS Synth. Biol.) to vary the tyrosine precursor levels (hereafter, named PCL1-"push"). To quickly test the new constructs, we conducted transient expression assay in N. benthamiana. The expression of the newly generated PAtLHB1B1- and PCL1-"push" constructs increased tyrosine level by >20- and >5-fold respectively compared to EV control. The expected YFP-tagged BvTyrAα size of ~73kDa was detected using anti-GFP in the protein extract from the infiltrated leaves expressing either the PAtLHB1B1- or PCL1-"push", confirming that inserted intron sequence was spliced out in N. benthamiana. Also, less amount of BvTyrAα protein was detected in the PCL1-"push" expressing leaves than the PAtLHB1B1-"push" expressing leaves, confirming that PCL1 has low expression level. We also conducted a coinfiltration assay using the "optimized pull" with the new "push" constructs. The maximum betacyanin production of 3.96 mg/gFW was achieved when the PAtLHB1B1-"push" was coexpressed with the "optimized pull", which was ~1.4-fold higher than the maximum betacyanin yield from the last year. Betalain levels in the leaves coexpressing the PCL1-"push" with the "optimized pull" did not show dramatic increase compared to the "optimized pull"-expressing leaves. Interestingly, in the leaves coexpressing the "optimized pull" with either the PAtLHB1B1- or PCL1-"push" showed significantly lower L-DOPA levels than the leaves coexpressing the "optimized pull" with the previous "push" construct. After confirming the functionality of the new constructs, we have been generating stable transgenic tobacco and soybean lines to test different responses on the same constructs from diverse plant chassis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Jung S. and Maeda H.A. (2024) Debottlenecking the L-DOPA 4,5-dioxygenase step with enhanced tyrosine supply boosts betalain production in Nicotiana benthamiana. Plant Physiol. in press.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
the Midwest ASPB 2024 Meeting from March 16th to 17th at Purdue University
Title: Debottlenecking the L-DOPA 4,5-dioxygenase step with enhanced tyrosine supply boosts betalain production in Nicotiana benthamiana
Authors: Soyoung Jung and Hiroshi A. Maeda
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Plant Metabolic Engineering Gordon Research Seminar, Barcelona, Spain, June 10-11, 2023
Title: Synthetic Biology to Enhance the Production of Tyrosine and Tyrosine-derived Compounds in Plants
Authors: Soyoung Jung and Hiroshi A. Maeda
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Plant Metabolic Engineering Gordon Research Conference, Barcelona, Spain, June 11 16, 2023
Title: Synthetic Biology to Enhance the Production of Tyrosine and Tyrosine-derived Compounds in Plants
Authors: Soyoung Jung and Hiroshi A. Maeda
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Gordon Research Conference on Plant Metabolic Engineering, Balcerona, Spain June 15, 2023
Title: Accelerating the conversion of CO2 into aromatic compounds in plants
Author/Presenter: Hiroshi A. Maeda
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Gordon Research Conference on Chloroplast Biotechnology, Ventura, California March 29, 2023
Title: Accelerating the conversion of CO2 into aromatic compounds in plants
Author/Presenter: Hiroshi A. Maeda
|
Progress 06/01/22 to 05/31/23
Outputs
Target Audience:The target audience of this plant synthetic biology project is broad, from agriculture industry that are interested in improving crop nutritional values (e.g. betalain pigments and vitamin E contents) as well as pharmaceutical industry that would like to improve the production of plant natural products (e.g. morphine and codeine, isoquinoline alkaloids derived from tyrosine) without compromising the overall crop yield. Toward this target audience in mind, we started experiments in model plants, such as Arabidopsis and Nicotiana, but then we are also carrying out studies in crops, such as tobacco, soybean, and sorghum, to test transformability of our metabolic engineering strategy. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for a graduate student, Soyoung Jung, together with a scientist, Marcos de Oliveira, to be trained in the interdisciplinary field of basic plant biochemistry and synthetic biology. We met weekly to discuss progress and next experimental plans. Soyoung and Marcos have also presented their results at the lab meeting, and Soyoung presented at the weekly PlantCMB seminar on campus, where graduate students and postdocs from UW campus present and exchange ideas. The PI Maeda also discussed with Soyoung and Marcos about their career goals at the beginning of the year using their individual developmental plan (IDP). We also participated in the Dane County Youth Apprenticeship Program in Biotechnology, which provides educational opportunities for high school students to support their scientific understanding and contributes to the continued success of the biotechnology industry. The program includes paidtraining in an industry or UW-Madison research lab. Students receive high school credit for their work experience (450 hours required per year)and for participation a specialized biotechnology course held weekly during the Academic Year at the BioPharmaceutical Technology Center Institute. Amy Yi from the local Verona high school joined our lab as a part of the Youth Apprenticeship program. Together with Soyoung Jung, she has been learning molecular biology and genetics tools to develop her project on characterizing a putative aromatic amino acid transporter in Arabidopsis. Amy has successfully isolated T-DNA knockout lines of OCT5 and other related genes and is currently making genetic crosses to make higher order mutants. How have the results been disseminated to communities of interest?Soyoung Jung attended the Annual Meeting of the Phytochemical Society of North America 2022 from July 24th to 28th to share our recent findings with the science community especially about plant synthetic biology and plant metabolic engineering. We also carried out in-person outreach events to disseminate our work to the general public and the importance of plant pigments and other metabolites to human well-being and society. At the UW Science Expedition, we carried out in-person outreach event called "BUILD-A-PLANT", together with four other laboratories of the PlantCMB group on UW campus to maximize our visibility. Among ~50 booths in the Health Science Learning Center with many medicine related topics, our plant outreach was one of the most popular booths due to hands-on activities where children can actually try themselves to learn. In the Maeda lab table, children carried out chromatography using different plant pigments, including betalain pigments, to learn about their properties and their importance in our society. This year, we also generated a "metabolic pathway" jigsaw puzzle for children to learn how different pigments are made from CO2 and sunlight energy through plant metabolic pathways. To communicate with a broader audience, we also carried out Science Night at Anana Elementary School in the city of Madison, where over 50% (51% in 2023) of enrolled students are from underrepresented minority groups. On the day of the event, we run four ~30 min hands-on activity with children and parents, using the plant pigments and jigsaw puzzle, to study different properties of plant pigments (metabolites) and explain how diverse metabolites produced in different plants are important for us. What do you plan to do during the next reporting period to accomplish the goals?Our data from this past year showed that DODA is a rate limiting step of the betalain biosynthetic pathway and by expressing additional DODA together with supplying additional precursor, betalain production was significantly increased in N. benthamiana. Also, our data from this past year showed that DODA expression even in the Ruby construct still needs to be optimized. Thus, we plan to generate a new optimized "pull" construct by including an additional transcriptional unit of DODA driven under the SlRbcS2 promoter to the Ruby construct. Furthermore, our data from this past year suggested that expressing the "push/pull" single construct might negatively affect the viability of Agrobacteria. It has been showed that including intron sequences in the middle of the coding sequence of transgene can prevent the marginal expression of the transgene in prokaryotes. Thus, we are planning to include intron2 sequence from the potato ST-LS1 gene for the "push" construct. Using new "push" and "pull" construct, we will quickly test the function of those constructs using transient infiltration system in N. benthamiana. Once their functionality is confirmed, we will then test these constructs through generation of stable transgenic lines in Arabidopsis, tobacco, and soybean. Our data suggested that once the downstream pathway is optimized, additional precursor supply can increase the production of target compound. Thus, we will investigate the optimized precursor levels for better production of target compound. To do this, we are planning to use different promoter with different strength, as well as use different deregulated enzymes, such as mutated AtTyrA2D241N_E179D (Lopez-Nieves et al., 2022) that we discovered recently and can increase tyrosine levels in planta, so that various "push" constructs can be tested with the optimized "pull" betalain construct. These new "push" constructs will be initially tested in Arabidopsis and Nicotiana, and promising combinations will be then introduced into tobacco and soybean.
Impacts
What was accomplished under these goals?
IMPACTS: Plants and crops provide promising platforms to efficiently produce diverse chemicals in a sustainable manner while absorbing CO2 from the atmosphere at the same time. However, our knowledge of implementing plant synthetic biology is highly limited. Over the past year, we have made significant progress in optimizing the betalain biosynthetic pathway to improve the production of downstream target compounds in plants. We identified a rate limiting step and investigated the impact of precursor supply to gain critical knowledge for successful implementation of plant synthetic biology for producing high levels of target specialized metabolites. Through our experiments using two different plant systems, Arabidopsis thaliana and Nicotiana benthamiana, we found that enhancing precursor supply and optimizing the target biosynthetic pathway by alleviating a rate limiting step, specifically the DODA enzyme, was critical for improving the production of betalain pigments, the major natural red color dye used in the US food industry. We also studied how plants respond to elevated tyrosine levels by using transgenic Arabidopsis lines and RNA-seq analysis, which allowed us to identify a potential vacuolar transporter affecting the availability of tyrosine, the starting material of betalain production. Our findings represent a significant step forward in plant synthetic biology and have practical implications for producing high-value compounds in plants for use in various industries. Specific Accomplishments for each objective: Objective 1) Contribution of tyrosine supply to betalain production using Arabidopsis. To better understand the interface of primary and specialized metabolism, we also investigated how plants respond to elevated supply of the precursor tyrosine. To this end, we performed RNA-seq analysis using two separate Arabidopsis transgenic lines overexpressing BvTyrAα (i.e., line #10 and #23) in comparison to ones expressing an empty vector (EV). We found that 44 and 8 genes were up- and down-regulated, respectively, in both BvTyrAα overexpressing lines compared to the EV control. Among those 44 upregulated genes, we found organic cation/carnitine transporter5 (AtOCT5, AT1G79410), which we hypothesized could function as a putative vacuolar tyrosine transporter to transport the excess tyrosine precursor into vacuole for sequestration. To test this hypothesis further, we have isolated Arabidopsis T-DNA knockout mutant lines of AtOCT5, crossed with the BvTyrAα overexpression lines, and will be analyzing metabolite profile of the crossed lines. By doing this, we expect to test the function of AtOCT5 and investigate better ways to efficiently use increased precursor levels for producing our target compounds in plants. Objective 2) Cross-species transformability of the betalains pathway reconstruction in various plants. In addition to the above work in Arabidopsis thaliana, this past year, we used Nicotiana benthamiana to further optimize the betalain biosynthetic pathway by identifying a rate limiting step and investigate the impact of precursor supply for better production of downstream target compound in planta. Previously, we hypothesized that additional precursor supply could better produce downstream target compounds in plants. To test this hypothesis, we generated DNA constructs overexpressing i) partially-deregulated tyrosine-synthesizing TyrA enzyme from Beta vulgaris (BvTyrAα, "push"), ii) three betalain biosynthetic genes ("pull"), iii) BvTyrAα and betalalin genes ("push/pull"), and iv) empty vector (EV) using golden gate assembly and transiently expressed those constructs in Nicotiana benthamiana using agroinfiltration method. However, we saw the unexpected results that lower pigmentation and lower transcript levels were observed in the leaves expressing the "push/pull" construct than those expressing the "pull" construct. These results suggested that the introduced betalain biosynthetic module have some limitations. This year, to test if the lower transcript levels in the "push/pull" construct can be rescued with the expression of p19 silencing suppressor, we conducted co-infiltration of the generated constructs (i.e., "push", "pull", "push/pull", or EV) with either p19 construct or EV. However, even with the p19 expression, we still saw lower pigmentation in the leaves when the "push/pull" construct is expressed than when the "pull" construct is expressed. Furthermore, we still saw lower transcript levels of all transgenes in the leaves expressing the "push/pull" construct than those expressing either the "push" or "pull" construct, even with the p19 expression. This result suggests that lower transcript levels in the "push/pull" construct might not be due to RNA silencing in plants. Next, we hypothesized that Agrobacterium harboring the "push/pull" construct could accumulate high levels of toxic intermediates (e.g., L-DOPA) because of a leaky expression of the DNA construct in Agrobacteria. Therefore, we tested if we could alleviate this potential problem by separating the BvTyrAα gene and the betalain genes into different Agrobacteria cells. We mixed each Agrobacterium cell harboring the "push" and the "pull" construct ("push"+"pull"), or the EV with the "pull" construct (EV+"pull"), and conducted co-infiltration. This time, we saw similar transcript levels of CYP76AD1 and DODA in the leaves co-expressing either "push"+"pull" or EV+"pull" constructs. However, we still saw the lower pigmentation when the "push" construct was co-expressed with the "pull" construct ("push"+"pull") than with the EV control (EV+"pull"). Metabolite analyses showed that higher levels of L-DOPA in the leaves co-expressing "push"+"pull" were not directed efficiently to betalain biosynthesis, resulting in lower betalain levels than in the leaves co-expressing the EV+"pull". This result suggests that the betalain biosynthetic pathway itself has a limitation in converting tyrosine and/or L-DOPA into betalains. To identify a rate limiting step(s) of betalain biosynthetic pathway, we generated additional constructs expressing each betalain biosynthetic gene (i.e., CYP76AD1, DODA, or cDOPA5GT) under the tomato rubisco small subunit 2 (SlRbcS2) promoter and co-expressed those constructs additionally with the previously generated constructs (i.e., "push", "pull", "push/pull", or EV) in N. benthamiana. We were able to produce higher betalains when additional DODA, encoding L-DOPA dioxygenase, was expressed than when other betalain biosynthetic genes were additionally expressed. The most interesting result here was that only when additional DODA construct was co-expressed, higher pigmentation was observed in the leaves co-expressing the "pull" and the "push" construct than the "pull" and EV construct, suggesting that DODA, which converts L-DOPA to betalamic acid, is a rate limiting step of the betalain biosynthetic pathway. We also tested the impact of precursor supply and additional DODA expression using the Ruby construct (He et al., 2020, DOI: 10.1038/s41438-020-00390-1), where three betalain genes were expressed under a single promoter, as our new pull construct. Here again, the highest pigmentation was observed when the Ruby construct was co-expressed with the "push" and additional DODA construct, followed by with EV and additional DODA construct. Our data indicate that enhancing precursor supply together with optimizing target biosynthetic pathway by alleviating a rate limiting step will be critical in successful implementation of plant synthetic biology to produce high levels of target specialized metabolites.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Maeda HA (2022) Deregulating aromatic amino acid biosynthesis. The Annual Meeting of the Phytochemical Society of North America, July 24th to 28th, Blacksburg, Virginia
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Jung S and Maeda HA (2022) Enhancing the production of tyrosine and tyrosine-derived compounds in plants using a synthetic biology approach. The Annual Meeting of the Phytochemical Society of North America, July 24th to 28th, Blacksburg, Virginia
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Progress 06/01/21 to 05/31/22
Outputs
Target Audience:The target audience of this plant synthetic biology project is broad, from agriculture industry that are interested in improving crop nutritional values (e.g. betalain pigments and vitamin E contents) as well as pharmaceutical industry that would like to improve the production of plant natural products (e.g. morphine and codeine, isoquinoline alkaloids derived from tyrosine) without compromising the overall crop yield. Toward this target audience in mind, we started experiments in model plants, such as Arabidopsis and Nicotiana, but then we are also carrying out studies in crops, such as tobacco, soybean, and sorghum, to test transformability of our metabolic engineering strategy. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for a graduate student, Soyoung Jung, together with a scientist, Marcos de Oliveira, to be trained in the interdisciplinary field of basic plant biochemistry and synthetic biology. We met weekly to discuss progress and next experimental plans. Soyoung and Marcos have also presented their results at the lab meeting. The PI Maeda also discussed with Soyoung and Marcos about their career goals at the beginning of the year using their individual developmental plan (IDP). How have the results been disseminated to communities of interest?Soyoung Jung will attend the Annual Meeting of the Phytochemical Society of North America 2022 from July 24th to 28th to share our recent findings with the science community especially about plant synthetic biology and plant metabolic engineering. We carried out both in-person and virtual outreach events to disseminate our work to the general public and the importance of plant pigments and other metabolites to human well-being and society. At the UW Science Expedition, we carried out in-person outreach event called "Odyssey of Plants", together with four other laboratories of the PlantCMB group on UW campus to maximize our visibility. Among ~50 booths in the Health Science Learning Center with many medicine related topics, our plant outreach was one of the most popular booths due to hands-on activities where children can actually try themselves to learn. In the Maeda lab table, children carried out different chemical reactions with plant pigments, including betalain pigments, to learn about their properties and their importance in our society. We also carried out virtual Science Night at Huegel Elementary School in the city of Madison, where over 50% of enrolled students are from underrepresented minority groups. It was initially planned for in-person events, but the organizer decided to switch to a virtual event at the last minute due to a surge in COVID cases in town. With the short notice, the entire lab worked together to prepare and deliver take home "Pigment Art" kits for ~150 students before the outreach event. On the day of the event, we run four ~30 min hands-on activity with children and parents to study different properties of plant pigments (metabolites) and explain how diverse metabolites produced in different plants are important for us. We also included petunia, mirabilis, and marigold seeds (with soil and pots) for them to grow and look at different pigments produced at home. What do you plan to do during the next reporting period to accomplish the goals?Our data from this past year showed that the betalain constructs that we generated were not able to pull the precursor towards betalain biosynthesis efficiently due to the low expression level of DODA transgene. Our data from this year using the Ruby construct suggests that elevated amount of precursor could produce higher level of betalains to the certain level, but the Ruby construct still needs to be optimized for higher betalain production. Thus, we plan to generate a new betalain construct with higher DODA expression for further experiments. For optimizing tyrosine supply to enhance production of downstream betalains, we plan to now test different TyrA enzymes with varied degrees of relaxed feedback inhibition. Our recent study using phylogeny-guided structure-function analysis of TyrA found additional key amino acid substitution E208D, other than previously known D222N substitution, in the active site of TyrA in core Caryophyllales for sensitivity to tyrosine inhibition. D241N and E179D are the substitutions equivalent to D222N and E208D respectively in Arabidopsis TyrA2 enzyme and the inhibition by tyrosine was not observed until 5 mM of tyrosine in mutated AtTyrA2D241N_E179D, whereas IC50 value of BvTyrAα was around 1 mM of tyrosine. By using this mutated enzyme, we can produce a much higher level of tyrosine in planta than using BvTyrAα. Thus, we will use this additional TyrA construct to test the correlation between tyrosine and betalain levels. After generating the overexpressing construct of BvTyrAα or AtTyrA2D241N_E179D for "push" construct, and the new betalain construct with higher DODA expression for "pull" construct, we will quickly test the function of those constructs using transient infiltration system into N. benthamiana. Once their functionality is confirmed (e.g., proper protein expression and accumulation of tyrosine and betalains), we will then test these constructs through generation of stable transgenic lines in Arabidopsis, tobacco, and soybean.
Impacts
What was accomplished under these goals?
Impact: During the second year of this four-year project, we tested the betalain constructs in different plant hosts including tobacco and soybean. We employed the Golden Gate assembly method to generate construct overexpressing i) partially-deregulated tyrosine-synthesizing TyrA enzyme from Beta vulgaris (BvTyrAα, "push"), ii) betalain biosynthetic genes ("pull"), iii) BvTyrAα and betalalin genes ("push and pull"), and iv) empty vector. The set of these four constructs were expressed in various plants and their metabolites and transgene expression levels were analyzed using LC-MS and qPCR, respectively. Through transient expression in Nicotiana benthamiana leaves, we found that the transgene expression of the second betalain pathway enzyme, L-DOPA dioxygenase (DODA), was low and thus the elevated level of tyrosine, due to BvTyrAα expression, cannot be effectively directed to betalain production. By expressing an additional DODA construct together with the BvTyrAα and betalain gene ("push and pull") construct, we were able to produce higher level of tyrosine and tyrosine-derived betalains in N. benthamaina. Our data suggests that it is important to enhance the precursor level together with careful reconstitution of target metabolic pathway by fine-tuning expression level of transgenes for efficient production of target metabolites in plants. Key outcomes: Based on our data from the previous reporting period (2020), we found that heterologous expression of betalain biosynthetic genes did not produce betalains in Arabidopsis. Thus, we hypothesized that choosing a proper plant chassis is important for successful reconstitution of complex biosynthetic pathway in host plant. This is likely because plants have diversified metabolism and regulatory systems mediating primary and specialized metabolism. To test if different plant hosts are more suitable for producing high level of betalains, we decided to use tobacco and soybean. Legumes, including soybean, have an additional cytosolic tyrosine biosynthetic pathway and therefore naturally have higher levels of tyrosine and tyrosine-derived compounds. Tobacco will be also a promising host, as successful reconstitution of betalain biosynthetic pathway have been reported mostly in Solanaceae, to which tobacco belongs. To test the different responses to the betalain constructs from diverse plant species, first we generated new betalain constructs for generating stable tobacco and soybean transgenic lines. Major differences between newly-generated betalain constructs and the previous ones were that we put a C-terminus tag for each transgene, so that we can directly assess the transgene expression levels by western blot. To quickly test the function of these newly generated constructs, we transiently expressed i) "push" (i.e., BvTyrAα driven by AtAct2 promoter) ii) "pull" (i.e., betalain construct) iii) "push/pull" (i.e., betalain construct with BvTyrAα) and iv) empty vector constructs into N. benthamiana. N. benthamiana leaves expressing the betalain constructs, both with and without BvTyrAα, clearly produced betalain pigments, confirming the new constructs are working. Unexpectedly, however, betalain pigments were produced lower in the leaves infiltrated with the "push/pull" construct than with the "pull" construct. To examine the metabolic responses to the newly generated constructs, we analyzed the infiltrated leaves for the levels of tyrosine and tyrosine-derived metabolites and transgene expressions using LC-MS and qPCR, respectively. We found that tyrosine was highly accumulated in the leaves infiltrated with the "push/pull" construct like we expected. Additionally, however, L-DOPA, an intermediate of the betalain pathway, was also highly accumulated in the plants with the "push/pull" construct. These results suggest that the high levels of tyrosine could produce elevated levels of L-DOPA, which was however not directed to betalain biosynthetic pathway efficiently due to the low expression level of the DODA transgene. To further test the constructs in stable transgenic plants, we generated stable tobacco lines with the same four constructs. Like Arabidopsis, stable transgenic tobacco with the "push" construct showed reticulated leaves and dwarf internode lengths due to high levels of tyrosine. However, unlike Arabidopsis not showing any betalain pigmentations with the "pull" construct, we were able to detect betalains in the transgenic tobacco with the "pull" construct. Despite our repeated attempts, we were not able to generate transgenic tobacco plants expressing the "push/pull" (i.e., betalain construct with BvTyrAα) construct, further suggesting that the toxicity of L-DOPA, or its derivatives, may negatively impact plant development. Soybean transgenic plants with the same four constructs are being generated at the Wisconsin Crop Innovation Center (WCIC) on campus and will be analyzed for transgene expression and different metabolite levels. To test if higher expression level of DODA will rescue this lower pigmentation phenotype, we generated new DODA constructs with either the SlRbcS2 or AtRbcS3 promoters, rubisco promoter from tomato and Arabidopsis, respectively, because these promoters have high expression levels based on prior reports. When we transiently expressed these additional DODA constructs together with the "push/pull" constructs (i.e., BvTyrAα with betalain genes), betalains were produced >70-fold and >25-fold higher in the leaves expressing the DODA constructs with SlRbcS2 and AtRbcS3 promoter, respectively, than the leaves without additional DODA construct. This data suggests that lower production of betalain in our "push/pull" construct was due to the low expression level of DODA and thus that increasing DODA expression will help improving betalain production for further experiments. Recently, He et al., 2020, reported that heterologous expression of the Ruby construct, where three betalain biosynthetic genes (CYP76AD1, DODA, cDOPA5GT) were expressed under a single promoter, can produce betalains in Arabidopsis and rice calli. Therefore, we obtained the Ruby construct from WCIC and carried out a similar experiment as above, to compare outcomes of two different betalain constructs. When we conducted co-infiltration assay of the Ruby construct under the constitutive GmScreamM8 promoter, elongation factor 1 promoter from soybean, with either BvTyrAα or empty vector into N. bentamiana, we were able to detect stronger pigmentation in the leaves co-infiltrated with the Ruby construct and BvTyrAα overexpressing construct than the leaves co-infiltrated with the empty vector. After analyzing tyrosine, L-DOPA, and betalain levels with LC-MS and spectrophotometer, respectively, the leaves co-infiltrated with the Ruby and BvTyrAα constructs showed >33- and >110-fold higher levels of tyrosine and L-DOPA, respectively, but only >1.4-fold higher betalain contents, than ones co-infiltrated the Ruby construct only. This data suggests that high level of precursor could enhance the betalain production to certain level; however, DODA activity is also limited in the Ruby construct and needs to be optimized for better production of betalains with enhanced precursor availability. Implication of the results: By understanding the impact of precursor supply together with reconstituting target metabolic pathway by fine-tuning the expression level of downstream betalain biosynthetic genes, we identified the limiting enzymatic step and were able to improve the production of betalain pigments in planta. This will provide a useful plant synthetic biology approach for successful production of target compounds by streamlining both supply and usage of amino acid precursors.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Lopez-Nieves S., El-Azaz J., Men Y., Holland C.K., Feng T., Brockington S.F., Jez J.M., Maeda HA* (2021) Two independently evolved natural mutations additively deregulate TyrA enzymes and boost tyrosine production in planta. Plant J 109, 844-855 https://doi.org/10.1111/tpj.15597
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Jung S. and Maeda HA (2022) Enhancing the production of tyrosine and tyrosine-derived compounds in plants using a synthetic biology approach. The Annual Meeting of the Phytochemical Society of North America, July 24th to 28th, Blacksburg, Virginia
|
Progress 06/01/20 to 05/31/21
Outputs
Target Audience:The target audience of this plant synthetic biology project is broad, from agriculture industry that are interested in improving crop nutritional values (e.g. betalain pigments and vitamin E contents) as well as pharmaceutical industry that would like to improve the production of plant natural products (e.g. morphine and codeine, isoquinoline alkaloids derived from tyrosine) without compromising the overall crop yield. Toward this target audience in mind, we will start experiments in model plants, such as Arabidopsis and Nicotiana, but then also carry out studies in crops, such as tobacco, soybean, and sorghum, to test transformability of our metabolic engineering strategy. Changes/Problems:Initially, it took an extra time to bring in the people to work on the project, partly due to the COVID-19 pandemic; however, the project is now up and running, and we should be able to make a steady progress going forward. What opportunities for training and professional development has the project provided?The project provided opportunities for a graduate student, Soyoung Jung, together with a scientist, Marcos de Oliveira, to be trained in the interdisciplinary field of basic plant biochemistry and synthetic biology. During the COVID pandemic, we met weekly to discuss progress and next experimental plans through online meetings. Soyoung and Marcos have also presented their results at the lab meeting. The PI Maeda also discussed with Soyoung and Marcos about their career goals at the beginning of the year using their individual developmental plan (IDP). How have the results been disseminated to communities of interest?Nothing to report. (We have focused on initiating the project during the first year and have not disseminated our findings to the community.) What do you plan to do during the next reporting period to accomplish the goals?As our preliminary data showed that the Betalain genes were not expressed well in Arabidopsis transgenic lines for some reasons, we plan to introduce both betalain cassettes into different model plants. It has been shown that stable heterologous production of betalains was conducted in a limited number of plant species, and most of those plant species were in Solanaceae family. Based on these reports, we first plan to introduce the betalain cassettes into Nicotiana tabacum, a model species of Solanaceae family. In most plant species, Tyr is synthesized within the plastids by the TyrA arogenate dehydrogenase (TyrAa/ADH) enzyme, whereas legumes have additional cytosolic Tyr biosynthetic pathway and the activity of PDH enzyme, which converts prephenate to HPP within the cytosolic Tyr biosynthetic pathway, is not feedback inhibited by Tyr. In our preliminary data, it has been confirmed that soybean, one of the legume plants, is more tolerant to high level of Tyr than most of other plants (unpublished result). Therefore, we plan to use soybean as another plant host to test the betalain cassettes. We confirmed that Arabidopsis can produce high level of Tyr, but somehow Arabidopsis wasn't able to utilize this Tyr for producing downstream products. Metabolic engineering in plants can have unexpected results like sequestration or chemical deactivation. Therefore, it might be informative to investigate how plants react transcriptionally and metabolically to high level of Tyr. To do this, we first plan to analyze transcriptional and metabolic changes in different Arabidopsis lines with high Tyr level including BvTyrAa overexpressing lines or the dhs2 mutant lines. We also plan to generate new construct with synthetic regulatory elements. Because there are a limited number of constitutive promoters used in plant engineering, there might have been limitations such as unintended pleiotropic effects or toxicity issues and the limited expression strength. To reconstitute the biosynthetic pathway of desired compounds in planta, we need to coordinate genes in the same biosynthetic pathway and fine-tune the expression level of each gene. Recently, Belcher et al. (2020 Nat Chem Biol 16, 857-865) developed and characterized a diverse set of synthetic promoters that can be coordinately activated by synthetic activators for the controlled modulation of transgene expression. We are planning to generate new constructs using some of the synthetic activator/promoter system for better coordination of our three or four transgenes. If successful, our study can provide useful examples for the use of these synthetic expression system by building plant production planforms with enhanced levels of Tyr and Tyr-derived compounds.
Impacts
What was accomplished under these goals?
Impact: During the first year of this four year project, we generated initial DNA constructs and expressed a series of biosynthetic enzymes to enhance production ("push") of tyrosine (Tyr) precursor and to utilize ("pull") Tyr for production of downstream natural products ("pull" i.e., betalain pigments). We were able to successfully increase the production of Tyr, and also heterologously produce betalain pigments in planta, providing promising starting point for further exploration and engineering, as detailed below. Key outcomes: We investigated how to enhance biosynthesis of tyrosine (Tyr) and its derivatives in planta. As Tyr plays crucial roles in plant growth and development, engineering Tyr biosynthesis in plant systems has not been well established, hindering our ability make use of bioactive compounds derived from Tyr. Our previous research showed that introducing Beta vulgaris TyrAa enzyme (BvTyrAa), which has relaxed sensitivity to Tyr feedback inhibition unlike other plant TyrAs, can increase the Tyr level in Arabidopsis while causing growth defect. We then examined if introducing the betalain biosynthetic pathway, which is one of the downstream pathways of Tyr, could enhance Tyr and/or Tyr derivative betalain production and could recover the plant growth defect caused by high Tyr level. We used the Golden Gate cloning method to generate DNA constructs for overexpressing a) betalain biosynthetic genes (CYP76AD1, cDOPA-5GT and DODA) and b) both BvTyrAa and the betalain genes. To further test if these constructs could function in planta to produce high levels of Tyr and Tyr-derivative betalain pigments, we transiently expressed both constructs, along with an empty vector negative control in Nicotiana benthamiana. The results showed that the N. benthamiana leaves infiltrated with the construct overexpressing betalain biosynthetic genes without BvTyrAa showed stronger betalain pigmentation than the leaves infiltrated with the construct overexpressing both BvTyrAa and betalain genes. To check the correlation between betalain pigment and the expression level of betalain genes in the infiltrated N. benthamiana leaves, we performed RT-qPCR. The result showed that BvTyrAa was only expressed in the leaves infiltrated with the construct both expressing BvTyrAa and betalain genes, but not in the leaves infiltrated with the construct expressing only betalain genes. However, the expression level of betalain genes were >10-20 fold higher in the leaves infiltrated with the construct expressing only betalain genes without BvTyrAa than ones with both BvTyrAa and betalain genes. From these transient expression experiments, we speculate that the expression of BvTyrAa might have negatively affected the viability of Agrobacteria cells leading to overall reduced efficiency of transient expression. Thus, the transient expression system might not be a suitable system to compare the effect of these constructs. To further test the functionality of these two constructs in planta, we generated transgenic Arabidopsis lines expressing either of the two constructs in the background of i) wild type (Col-0), ii) BvTryAa overexpressors, and iii) the dhs2 A4 mutant that accumulates all aromatic amino acids including Tyr due to a point mutation in the first enzyme of the upstream shikimate pathway, 3-deoxy-D-arabinoheputulosonate 7-phosphate synthase 2 enzyme (DHS2, unpublished data in our laboratory). Our Initial results showed that some T1 Col-0 and dhs2 A4 mutant lines transformed with the betalain cassette containing 35S::BvTyrAa showed reticulate leaf and growth defect phenotypes but no Betalain production could be visually identified. Tyrosine levels of T1 plants by HPLC showed a positive correlation between high level of Tyr and development of reticulate leaves and growth defect. This result suggests that the cassettes were integrated well into different Arabidopsis lines, but somehow high level of tyrosine cannot be utilized for producing downstream products in Arabidopsis. Implication of the results: While the project is still in progress, establishing the metabolic engineering system in a model system like Arabidopsis will allow us to apply the system to other plant species. We are indeed planning to express some of the successful constructs in crops, such as soybean and sorghum, which can take advantage of their large biomass for efficient production of Tyr and its derived compounds.
Publications
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