Progress 09/01/16 to 08/31/21
Outputs
Target Audience:Scientific community, international conferences (only for the reporting period, 2020-2021): 2020 · Invited talk, MSU SciComm, Collisions between Science and Art (February 2020) · Invited talk Gordon Research Conference (postponed due to COVID-19) 2021 · Invited talk at Calyx Inc. Minnesota (February 2021) · Invited talk New England Biolabs Inc. (August 2021) · Invited talk Truman State University (September 2021) · Invited talk, University of Minnesota, Center for Precision Plant Genomics (October 2021) · Invited talk, BASF Plant Sciences, Raleigh, NC (October 2021) · Invited talk, University of Michigan (October 2021) Special focus of CANR, MSU communications and NatSci Connections on our SPG collaboration with focus on new libraries of anti-microbial compounds (January 24, January 31, and Winter 2020) https://www.canr.msu.edu/news/plant-molecules-show-promise-in-priming-the-antibiotic-pipeline https://www.canr.msu.edu/news/new-library-of-plant-molecules-established-in-the-fight-to-discover-new-antibiotics https://www.bluetoad.com/publication/?m=40206&i=685440&p=12 · COVID-19 had a strong impact on our typical in-person outreach activities in 2020, but we expect to resume our collaborations and activities as soon as possible. Changes/Problems:Millet and Brachypodium were not found to be genetically tractable. Transformation of the grass species Sorghum is in the planning stage (DOE funded), while a new NSF funded project aims at generating a minimal plant genome in tomato, depleted of terpene background, and transformation of that chassis with high value pathways. What opportunities for training and professional development has the project provided?The iGEM undergraduate research experience is an ongoing activity in 2021 (since 2016 at MSU). This program is aimed at mentoring and training of a team of undergraduate students representing MSU at the annual Synthetic Biology World Championship (iGEM, internationally genetically engineered machine) in Boston. All 2021 activities conform to MSU guidelines during COVID-19. Former postdoc Rock Du has accepted a faculty position at the University of Hawaii. Former postdoc Sean Johnson now is employed at New England Biolabs, current postdoc Wajid Bhat has received a job offer from Ginkgo Bioworks and also has an offer for a faculty position (India). Former postdoc Aparajita Banerjee has accepted a postdoc position at the University of Delaware. PhD student Emily Lanier has a job offer from an IP consulting company. How have the results been disseminated to communities of interest?Communication and in-person outreach, public engagement in this period was halted due to COVID-19. An exploratory meeting with representatives of the Anishinabeg is planned for 11/04/2021. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Reporting period 2020-2021, please see previous NIFA reports for details for 2016-2019. 1. Engineering of biotechnological chassis systems for biosustainable production 1.1 Transfer of functionally characterized terpenoid biosynthetic routes to Physcomitrella The last experiment for the engineering of Physcomitrella turned out challenging, but this will be completed in 2021, with a manuscript in preparation (PhD student Davis Mathieu). The search for additional, possibly better suited chassis systems in 2021 led to engineering of the microalgae Nannochloropsis (experimental part finished, paper in preparation, Dr. Wajid Bhat, in collaboration with Asst. Prof. Rock Du (former lab member, now at University of Hawaii)), poplar (PhD student Jake Bibik, in collaboration with Christos Maravelis, Princeton, technoeconomic analysis pending), where we accomplished yields of the high value triterpene squalene close to theoretical commercial competitiveness and the oilseed plant camelina (experimental part finished, paper in preparation, Dr. Wajid Bhat). 1.2 Transformation of model grasses Brachypodium and Millet with pilot terpenoid biosynthetic routes and testing of production capacities and feasibility of the strategy Transformation efficiency of Brachypodium was found very low, while the published procedure for Millet was found not reproducible by our team and other labs in the field. These two species were consequently abandoned. We have recently been awarded a DOE Emerging Opportunity Award to target specific tissues for terpene production and secretion from the hairy roots of the alternative monocot plant species Sorghum. Generation of constructs is underway, the transformation procedure is established (Dr. Trine Andersen, in collaboration with the Wisconsin Crop Innovation Cente). 1.3 Discovery of new pathways to high-value terpenoids in plant species selected for their unique accumulation (in cooperation with Buell) Our recent findings in snowberry (Chiococca alba), beautyberry (Callicarpa americana) and Kratom (Mitragyna speciosa) are published independently in three manuscripts. Specifically, our discoveries of a large gene cluster, conserved across all investigated members of the mint family have inspired new research on its dynamic evolution and function (PhD students Abby Bryson, Emily Lanier, manuscript in preparation). 1.4 Complementing the existing terpenoid biosynthetic toolbox with new functional parts to expand our biosynthetically accessible chemical space We have achieved production of over twenty novel, non-natural diterpenes using our biosynthetic toolbox in combination with synthetic substrates. The new NMR equipment at the department of Chemistry has dramatically accellerated their structural elucidation. A manuscript is in preparation (PhD student Garret Miller, in collaboration with Dr. Edmund Ellsworth, MSU Pharmacology and Toxicology). Notably, a biosynthetic library of terpene products has resulted in active and specific hits in a screen for anti-cancer activity (PhD student Garret Miller, collaboration with Dr. Jamie Bernard, MSU Pharm&Tox). Several strategies for scaling of production are currently ongoing to meet the needs for follow-up screening and testing (graduate student Duncan Boren). 1.5 Testing of terpenoid biosynthesis in context of modulated lipid biosynthesis in the transient Nicotiana benthamiana system and selection of optimal pathways and parts All parts and control of their subcellular localization were finished. Intriguingly, co-engineering of lipid droplets and terpene biosynthetic pathways alleviated the impact on photosynthesis significantly. A first manuscript draft is in editing (PhD student Jake Bibik, photosynthesis experiments in collaboration with Dr. Tom Sharkey's lab, subcellular localization in collaboration with Dr. Peter Lundquists lab (MSU)). 2. Adding value to the most recent generation of biofuel crops 2.1 Integrating the knowledge from 1.2 and 1.5 with engineered lines of grasses for enhanced production of lipids (in cooperation with Benning) those lines are transformed with selected terpenoid pathways to evaluate their potential application in production. See point 1.1 and 1.2 for details and our progress with engineering of biofuel species. The work in poplar was successful, a revised strategy is being deployed in Sorghum. 2.2 Test if the entry step into the toxic phorbol esters, we have identified in paper spurge can be targeted by CRISPR and if so, generation of toxin-free lines After a paper from another lab published results in phorbol ester reduction using RNAi, this research project was abandoned. 2.3 Determination of the resistance to pests or pathogens of the engineered lines (cooperating team will have to be identified) The Scientific leadergroup of the GLBRC strongly discouraged field trials with our engineered elite lines producing squalene. We are currently investigating other sources of support and funding. 3. Advancing our knowledge on the interaction of plants with their environment to guide future improvement of fitness and protection 3.1 Establish plant-endophyte interaction in the system Mortierella/Physcomitrella or Mortierella/Indian coleus The plant endophyte system is established and under phenotypic and transcriptomic investigation, with a manuscript nearing completion (PhD student Davis Mathieu, part of our NSF Biodimensions collaborative project with the lab of Dr. Greg Bonito (MSU)). This project led to additional funding to investigate the Sorghum root - microbiome interaction. 3.2 Evaluate a potential impact on plant fitness and terpenoid production While the stably engineered poplar lines showed no visible, or phenotypic impact caused by the production of the terpene squalene, an in depth investigation of the photosynthetic activity showed a reduction, which could be rescued by co-engineering of lipid droplets.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Lau KH, Bhat WW, Hamilton JP, Wood JC, Vaillancourt B, Wiegert-Rininger K, Newton L, Hamberger B, Holmes D, Hamberger B*, Buell CR (2020)
Genome assembly of Chiococca alba uncovers key enzymes involved in the biosynthesis of unusual terpenoids.
DNA Res. 2020 Jun 1;27(3):dsaa013. doi: 10.1093/dnares/dsaa013. PMID: 32642754
*Shared corresponding authorship.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Hamilton JP, Godden GT, Lanier E, Bhat WW, Kinser TJ, Vaillancourt B, Wang H, Wood JC, Jiang J, Soltis PS, Soltis DE, Hamberger B, Buell CR (2020)
Generation of a chromosome-scale genome assembly of the insect-repellent terpenoid-producing Lamiaceae species, Callicarpa americana.
Gigascience. 2020 Sep 1;9(9):giaa093. doi: 10.1093/gigascience/giaa093. PMID: 32893861
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Miller GP, Bhat WW, Lanier ER, Johnson SR, Mathieu DT, Hamberger B. (2020)
The biosynthesis of the anti-microbial diterpenoid leubethanol in Leucophyllum frutescens proceeds via an all-cis prenyl intermediate.
Plant J. 2020 Aug 10. doi: 10.1111/tpj.14957. PMID: 32777127
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Brose J, Lau KH, Dang TTT, Hamilton JP, Martins LDV, Hamberger B, Hamberger B, Jiang J, O'Connor SE, Buell CR. The Mitragyna speciosa (Kratom) Genome: A resource for data-mining potent pharmaceuticals that impact human health. (2021) G3 (Bethesda). doi: 10.1093/g3journal/jkab058. PMID: 33677570
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ntana F, Bhat WW, Johnson SR, J�rgensen HJL, Collinge DB, Jensen B, et al. A Sesquiterpene Synthase from the Endophytic Fungus Serendipita indica Catalyzes Formation of Viridiflorol. Biomol. (2021) 11(6), 898. https://doi.org/10.3390/biom11060898
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Please see previous reports and NCBI MyBibliography: https://goo.gl/HLCcf4 (59 papers); patents at patents.google.com (search terms Hamberger AND Terpene) for publications prior to 2020.
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience: Scientific community, international conferences: Invited Speaker, Inaugural Talk, Postgraduate Students Seminar Program, CICY, Merida (Mexico), November 4-6 (Centro de Investigación Científica de Yucatán) Presentations by team members Jacob D. Bibik, Björn Hamberger (September 2019, oral presentation), "Synthetic Biology Approaches Establish the Foundation for Sustainable Production of High Value Terpenoids". Michigan State University, CMB & GGS Research Forum. East Lansing, Michigan, USA Jacob D. Bibik, Aparajita Banerjee,Björn Hamberger (February 2020, poster presentation)"Pathway Engineering and Re-targeting Boosts Production of High-Value Bioproducts in Plants". DOE - Genomic Sciences Program Annual PI Meeting. Washington, D.C., USA. ?Jacob D. Bibik, Aparajita Banerjee, Ka'Shawn Robertson,Björn Hamberger (May 2020, oral presentation)"Engineering plants as a platform for terpenoid production".Great Lakes Bioenergy Research Center Annual Science Meeting. Virtual. Broader public community MSU Science Communication organization's first live Science Art show held October 19th(FRIB facility). Presenters graduate student Davis Mathieu and Björn Hamberger with the last public showing for "Fog of Dawn". Audience: approximately 600 visitors. Comments: Due to COVID-19 the Fascination of Plants Day 2020 collaboration with the Broad Museum was cancelled. I am directly involved in the organization of the annual meetings of the Phytochemical Society of North America. The meeting in 2020 was postponed to 2021. Changes/Problems:All current challenges beyond COVID-19 are addressed in the 'goals accomplished' text. No further contingencies are needed. What opportunities for training and professional development has the project provided? The iGEM undergraduate research experience is an ongoing activityin 2020. This program is aimed at mentoring and training of a team of undergraduate students representing MSU at the annual Synthetic Biology World Championship (iGEM, internationally genetically engineered machine) in Boston. All 2020 activities conform to MSU guidelines during COVID-19. How have the results been disseminated to communities of interest?Communication and in-person outreach,public engagement in this period was halted due to COVID-19. A NatSci communication regarding Science during COVID-19 and microbial resistance is in production. What do you plan to do during the next reporting period to accomplish the goals? We are on track and no immediate challenges beyond COVID-19 are recognized, which could impede progress. The NIH proposal was positively evaluated, but ultimately not funded. We are gathering additional data for resubmission. This proposal served as template for a large center-type NIH application (NIH RM-1); Hamberger co-director.
Impacts
What was accomplished under these goals?
1. Engineering of biotechnological chassis systems for biosustainable production 1.1 Transfer of functionally characterized terpenoid biosynthetic routes to Physcomitrella We could demonstrate stable expression of our LTR integrated reporter lines for 18 months. Replacement of three representative LTR elements with diterpene biosynthetic pathways is underway. Despite stableproduction capacity in Physcomitrella, yields have so far remained low. To further mitigate this risk, the algae Nannochloropsis oceanica was added as chassis species. We could demonstrate successful engineering with a synthetic pathway to the high-value diterpene Casbene. A paper is in preparation. 1.2 Transformation of monocotyledons: Transformation of model grasses Brachypodium and Millet with pilot terpenoid biosynthetic routes and testing of production capacities and feasibility of the strategy Brachypodium transformation remained at a low efficiency. However, several lines were recovered which now constitutively produce the sesquiterpene caryophyllene. Our efforts in transforming Millet were unsuccessful, both earlier reports describing transformation of Millet were not reproduced by the community and several other teams have now terminated their Millet platforms as result. Replacing Millet with Sorghum is currently underway in our lab. The Sorghum set of terpene metabolic genes was identified, curated and mapped to the most gecent genome. In collaboration with John Mullet (Texas A&M) we also performed expression analysis of all genes and mapped a specific subset to the root. Constructs for Sorghum transformation are in preparation. 1.3 Discovery of new pathways to high-value terpenoids in plant species selected for their unique accumulation (in cooperation with Buell) We published the complete elucidation of the pathway to the anti-microbial diterpene alcohol Leubethanol in August 2020, with NIFA support acknowledged. We discovered a route to the high-value anti-fungal sesquiterpene alcohol Viridiflorol in the fungal species Serendipita indica. A patent has been filed (manuscriptin revision). With funding from other sources, we published the following manuscripts describing discovery of terpene related pathways in teak (Tectona grandis), snowberry (Chicocca alba) and beautyberry (Callicarpa americana)(no NIFA suport). Zhao D, et al., Hamberger B, Buell CR. A chromosomal-scale genome assembly of Tectona grandis (...). Gigascience. 2019. doi: 10.1093/gigascience/giz005. Lau KH, et al.,Hamberger B, Holmes D, Hamberger B*, Buell CR (2020) Genome assembly of Chiococca alba (...). DNA Res. 2020 Jun 1;27(3):dsaa013. doi: 10.1093/dnares/dsaa013. Hamilton JP, et al.Hamberger B, Buell CR (2020) Generation of a chromosome-scale genome assembly of (...) Callicarpa americana. Gigascience. 2020 Sep 1;9(9):giaa093. doi: 10.1093/gigascience/giaa093. 1.4 Complementing the existing terpenoid biosynthetic toolbox with new functional parts to expand our biosynthetically accessible chemical space (now integrated with 1.5) 1.5 Testing of terpenoid biosynthesis in context of modulated lipid biosynthesis in the transient Nicotiana benthamiana system and selection of optimal pathways and parts Lipid droplet scaffolding and engineering: Compartmentalization of pathways and products can enable storage of products and reduce interference of native pathways to unwanted modification of desired products, increasing overall yields. We report two engineering strategies developed to enhance squalene production in plants: (i) pathway re-targeting to plastids and (ii) scaffolding of pathwayson lipid droplets.The squalene pathway was optimized at three key steps through screening of diverse, heterologous enzymes resulting in increased yields. The highest yielding combination consisted ofArabidopsis thalianafarnesyl diphosphate synthase, a truncatedMortierella alpinasqualene synthase,Coleus forskohliideoxy-xylulose-phosphate synthaseand a truncatedEuphorbia lathyrishydroxy-methylglutaryl CoA reductaseto boost the plastidial MEP and cytosolic MVA pathways, respectively. The optimized pathways were combined with theA. thalianatranscription factor WRI1 and theNannochloropsis oceanicaLDSPto induce formation of cytosolic lipid droplets and to scaffold and to scaffold FDPS and SQS LDSP fusions on lipid droplets, which further increased yields. This strategy demonstrated high modularity in fusion combinations, which allows for flexible construct design and efficient expression of up to five genes, yielding up to 0.78% (cytosol LD) and 0.4% (plastid) of squalene in leaf fresh weight, which meet recent criteria of economic competitiveness. Effects on photosynthesis: (The following are results of a collaboration with the team of Tom Sharkey)Three variations of the optimized squalene strategies are being used to measure their effects on plant physiology: (i) plastid re-targeted pathway, (ii) cytosolic lipid droplet scaffolding, (iii) and the LDSP fusion proteins re-targeted to plastids. Re-targeting the LDSP fusion proteins may enable formation of lipid droplets within plastids or membrane association of the pathway, sequestering squalene. Expression of the squalene pathway in plastids led to a significant decrease in photosynthesis five days after infiltration.While a reduction in stomatal conductance was observed in all three treatments, internal [CO2] was unchanged indicating that stomata are not responsible for the changes in photosynthetic rate.Interestingly, leavesexpressing the squalene pathway in plastids fused to the LDSP gene significantly recovered to higher rates of photosynthesis (45%) than leaves without the LDSP fusion. The observed increase in photosynthetic rate at this time point co-occurs with a relative reduction in squalene production. In contrast, our calculations show that only 0.1 to 0.2% of the total carbon fixed during photosynthesis was used for squalene synthesis. This indicates (i) significant headroom remaining in the biosynthetic capacity for production of terpene bioproducts, and (ii) potential benefits of LDSP, including formation of plastid-based lipid droplets, i.e. plastoglobules and lipid, or membrane association of the pathway. The effect of the LDSP on the chloroplast and squalene pathway is currently under investigation. Manuscript in preparation. 2. Adding value to the most recent generation of biofuel crops 2.1 Integrating the knowledge from 1.2 and 1.5 with engineered lines of grasses for enhanced production of lipids (in cooperation with Benning) those lines are transformed with selected terpenoid pathways to evaluate their potential application in production. While constructs with the LD parts proved lethal at the rooting stage of poplar regeneration, a panel of stably plastid engineered poplar was transferred to soil. To address the issues with the impact on poplar viability, a second generation of poplar constructs is currently in preparation, replacing the constitutice CaMV35S promoter with poplar leaf specific elements. 2.2 Test if the entry step into the toxic phorbol esters, we have identified in paper spurge can be targeted by CRISPR and if so, generation of toxin-free lines Specific subgoal abandoned. VIGS vectors were receiced (Sarah O'Connor, Germany). 2.3 Determination of the resistance to pests or pathogens of the engineered lines (cooperating team will have to be identified) 3. Advancing our knowledge on the interaction of plants with their environment to guide future improvement of fitness and protection 3.1 Establish plant-endophyte interaction in the system Mortierella/Physcomitrella or Mortierella/Indian coleus 3.2 Evaluate a potential impact on plant fitness and terpenoid production Goal delayed due to COVID-19.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Zhao D, Hamilton JP, Bhat WW, Johnson SR, Godden GT, Kinser TJ, Boachon B, Dudareva N, Soltis DE, Soltis PS, Hamberger B, Buell CR. A chromosomal-scale genome assembly of Johnson SR, Bhat WW, Sadre R, Miller GP, Garcia AS, Hamberger B. (2019)
Promiscuous terpene synthases from Prunella vulgaris highlight the importance of substrate and compartment switching in terpene synthase evolution.
The New Phytologist. 2019; doi: 10.1111/nph.15778. PMID: 30843212
- Type:
Book Chapters
Status:
Published
Year Published:
2020
Citation:
Britta Hamberger, Macaire Man Saint Yuen, Emmanuel Buschiazzo, Claire Cullis, Agnes Yuen, Carol Ritland, J�rg Bohlmann and Bj�rn Hamberger. An intact, but dormant LTR retrotransposon defines a moderately-sized family in white spruce (Picea glauca). Springer Nature, The Spruce Genome. Eds. Ilga Mercedes Porth and Amanda De La Torre.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Miller GP, Bhat WW, Lanier ER, Johnson SR, Mathieu DT, Hamberger B. (2020)
The biosynthesis of the anti-microbial diterpenoid leubethanol in Leucophyllum frutescens proceeds via an all-cis prenyl intermediate.
Plant J. 2020 Aug 10. doi: 10.1111/tpj.14957. PMID: 32777127
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Scientific community, international conferences: 2018 Invited talk, 4th International Symposium on Functional Food and Plant Metabolism, Shanghai, China, December 15-17 2019 Invited talk, At the Forefront of Plant Research, Barcelona (Spain) May 6-8 (Centre for Research in Agricultural Genomics, CRAG) Selected talk, The 14th International TerpNet Meeting, Halle (Germany) August 26-30 (Martin-Luther-Universität) Presentations by team members: Jake Bibik, "Improved squalene production in plants through pathway optimization and compartmentalization" Annual Meeting (July 20 -24, Eastern Tennessee State University, Johnson City, TN, USA) and Gordon Research Conference for Plant Metabolic Engineering, Metabolic Engineering in Plants: From Design Concepts to Applications, June 16 - 21, 2019 (Lucca (Barga), IT). Garret Miller, "Structural and Sequential Analysis of Functionally Divergent Terpene Synthases in Prunella vulgaris", Synthetic Biology, Evolution, Engineering, and Design (SEED); NYC, June 23, 2019. Davis Mathieu, Genetics Mini-Symposium (MSU, May 7th, 2019) -High-Throughput Phenomic Analysis ofPhyscomitrella patens; ASBMB (MSU, May 7th, 2019) -High-Throughput Phenomic Analysis ofPhyscomitrella patens; ASPB-Annual Meeting San Jose, CA (Aug 3-7th, 2019) -High-Throughput Phenomic Analysis ofPhyscomitrella patens; ASPB- Synthetic Biology San Jose, CA (Aug 7 - 9th, 2019) -High-Throughput Phenomic Analysis ofPhyscomitrella patens;Flexible Fluorescent Tagging to Elucidate Ancestral Plant Traits. Press releases/public outreach: 2018 Synthetic biology may hold key to answering some of the most difficult science questions: https://www.canr.msu.edu/news/synthetic-biology-may-hold-key-to-answering-some-of-the-most-difficult-science-questions 2019 Harnessing Synthetic Biology to produce terpenoids and oil in plants: https://msutoday.msu.edu/news/2019/harnessing-synthetic-biology-to-produce-terpenoids-oil-in-plants/ The third fascination of plants day (FoPD@MSU) is our first collaboration with the MSU Broad Museum: https://msutoday.msu.edu/360/2019/anne-sophie-bohrer-for-the-love-of-plants/ TALK: THE INTERSECTION: THE SCIENCE OF NATURE AND TECHNOLOGY (Museum of Contemporary Art, Detroit) https://mocadetroit.org/events-and-education/talk-intersection-science-nature-technology/ The Collision of Art and Science (Detroit Science Gallery) https://detroit.sciencegallery.com/events/collision-art-and-science Coverage of collaborative project between our team and artists H. Dewey-Hagborg and P. A. Lewis: https://broadmuseum.msu.edu/exhibitions/spirit-molecule MSU today, Faculty Voice, coverage of the Detroit Science Gallery Exhibition piece "Fog of Dawn" https://msutoday.msu.edu/360/2019/bjrn-hamberger-when-science-and-art-come-together/ The third Fascination of Plants Day at MSU 2019 was the first collaboration between our MSU initiative, the East Lansing Art Festival and the Broad Museum. Audience: 330 participants. Our project proposal for the Detroit Science Gallery "Fog of Dawn" was selected for a contribution to the Exhibition "Depth" in June to August 2019 (lead-PI Hamberger, co-PIs Sarah Evans, Greg Bonito). Audience: approximately 30,000 visitors. In collaboration with the Steven L. Bridges, Associate Curator, Eli and Edythe Broad Art Museum and artists Heather Dewey-Hagborg (https://deweyhagborg.com/) and Philipp Andrew Lewis (http://www.phillipandrewlewis.com/), our team was involved with an exhibition from June-September 2019 featuring transgenic and engineered moss. Science Cafes and Pubs (Science Gallery Detroit) and East Lansing Science Fair: Collisions Between Science and Art at the Broad Museum Art Lab. Björn Hamberger, sneak preview of "Fog of Dawn", followed by dialogue with the public. Moderated by Antajuan Scott, Head of Programming and Mark Sullivan, Creative Director, Detroit Science Gallery. April 7, 2019. Audience: approximately 70 participants. The Intersection: The Science of Nature and Technology. Using the nature-based artwork and themes presented in the exhibition Parallels and Peripheries as a starting point, this talk will explore how artists and scientists utilize nature to push the boundaries of our understanding of technologies within the contemporary art world. Moderated by Larry Ossei-Mensah, Susanne Feld Hilberry, Senior Curators at Museum of Contemporary Art Detroit, and featuring artists Ash Arder and Susan Goethel Campbell, in conversation with Michigan State University Assistant Professor Bjoern Hamberger. April 13, 2019. Audience: approximately 80 participants. MSU Science Communication organization's first live Science Art show held October 19th (FRIB facility) Changes/Problems:Postdoc Aparajita Banerjee will leave the team in Fall 2019. We were excited to have just hired Dr. Trine Andersen with an excellent background in terpenoid research to replace Aparajita. Trine started in september 2019. We also invited applications for a postdoctoral position for our collaborative project with BASF. Review of applications is underway. What opportunities for training and professional development has the project provided?In 2019, the NIH T32 Pre-doctoral Biotechnology Training Grant "Plant Biotechnology for Health & Sustainability" (NIH T32, PBHS), where I serveas Executive Committee member, was renewed. The iGEM undergraduate research experience is an ongoing activity with, in 2019, a grwoing number of faculty mentors. This program is aimed atmentoring and training of a team of undergraduate students representing MSU at the annual Synthetic Biology World Championship (iGEM, internationally genetically engineered machine) in Boston. The first iGEM team at MSU in 2016 received a Bronze medal (2017, Silver medal, 2018 Bronze). MSU iGEM 2019 in preparation for the Competition in Boston (November 2019). Outreach activities of the iGEM teams included lectures, demonstrations, interactive activities, public surveys at, for instance, Impression 5 Science Center, the East Lansing Public Library, the Capital Area Public Library, the Michigan Science Center and during organized North American Upper Midwest Meet-ups at MSU. How have the results been disseminated to communities of interest?Communication of MSU Plant Research to the public: We increasingly understand the critical importance of efficient communication of our research to a broad public audience. The Fascination of Plants Day is a global event under the umbrella of the European Plant Science Organization, giving exactly this opportunity. In 2017, the first year the event took place in the US, MSU spearheaded the initiative. I initiated the event and together with two postdocs, who took on the roles of event manager (Anne-Sophie Bohrer) and volunteer coordinator (Aparajita Banerjee), financial support was gathered from the Office of the Vice President for Research and Graduate Studies, the College of Natural Sciences, the MSU-DOE Plant Research Lab, the Biochemistry and Molecular Biology Program and the Department of Plant Biology. This initiative has reached with its third year several milestones, including increased visibility through a collabroation with the MSU Broad Museum and the East Lansing Arts Festival. Several hundred visitors were engaged in the 2019 MSU Fascination of Plants Day.We are planning of building on and expandingthis effort in 2020. The third fascination of plants day (FoPD@MSU) is our first collaboration with the MSU Broad Museum: https://msutoday.msu.edu/360/2019/anne-sophie-bohrer-for-the-love-of-plants/ Additional outreach activities include public talks and discussions (Museum of Contemoprary Art, Detroit; Detroit Science Gallery; MSU Broad Museum). TALK: THE INTERSECTION: THE SCIENCE OF NATURE AND TECHNOLOGY (Museum of Contemporary Art, Detroit) https://mocadetroit.org/events-and-education/talk-intersection-science-nature-technology/ The Collision of Art and Science (Detroit Science Gallery) https://detroit.sciencegallery.com/events/collision-art-and-science Coverage of collaborative project between our team and artists H. Dewey-Hagborg and P. A. Lewis: https://broadmuseum.msu.edu/exhibitions/spirit-molecule MSU today, Faculty Voice, coverage of the Detroit Science Gallery Exhibition piece "Fog of Dawn" https://msutoday.msu.edu/360/2019/bjrn-hamberger-when-science-and-art-come-together/ Target audience, scientific community: Three talks at national and international conferences were great opportunities to discuss and disseminate latest research findings (Hamberger). Direct involvement in the organization of the relevant meetings in 2020 will ensure continuity and an increasing network (International TerpNet Conference, Phytochemical Society of North America). Members of the team have in 2019 delivered 9 contributions and presentations. Target audience, potential industrial partners: With the successful agreement with BASF in place, no further partners were approached in 2019. What do you plan to do during the next reporting period to accomplish the goals?We are on track and no immediate challenges are recognized, which could impede progress. Being awarded permanent residency opened opprotunities within NIH. Aa New Investigator (Hamberger) grant proposal with the title "Biosynthesis of chemically diversified non-natural terpene products" was submitted for consideration under the NIH Research Grant Program (Focused Technology Research and Development) with PA number PAR-19-253. Key senior personnel: Rick Neubig, Edmund Ellsworth (MSU, Pharmacology & Toxicology). Fueled by discoveried in the Lamiaceae family, we have initated a funding application to NSF (PI, Hamberger, co-PI, Natalia Dudareva (Purdue University)). Currently pending is a large Center Grant (NSF, Directors Bob Hausinger, Christoph Contag; Hamberger lead of one of four thrust areas and one of eight research nodes).
Impacts
What was accomplished under these goals?
1. Engineering of biotechnological chassis systems for biosustainable production 1.1 Transfer of functionally characterized terpenoid biosynthetic routes to Physcomitrella We earlier described the successful modular engineering of diterpene biosynthetic pathways in Physcomitrella (Banerjee et al., 2018). This work led to discovery of loci created by Long Terminal Repeat Retrotransposons as potential integrative locations for targeted genome engineering. During the last year, we have generated a library of Physcomitrella lines, targeted for the LTR loci with a fluorescent reporter and we could show significant differences in transgene expression. These lines are now used for transformation with diterpene biosynthetic pathways to test whether these loci can modulate expression, and formation of diterpene products. 1.2 Transformation of model grasses Brachypodium and Millet with pilot terpenoid biosynthetic routes and testing of production capacities and feasibility of the strategy Genotyping of Brachypodium lines showed that several constructs were successfully integrated in both wild-type and lines engineered for lipid droplet formation. Segregation analyses and chemotyping are underway and should be finished in early 2020. 1.3 Discovery of new pathways to high-value terpenoids in plant species selected for their unique accumulation (in cooperation with Buell) The manuscript describing the discovery and engineering of new pathways in the Lamiaceae family has been published (Johnson et al., 2019). A related patent has been filed (October 2019, US2019/044887). This work consequently led to the specific investigation of the Lamiaceae species common selfheal (Prunella vulgaris), selected for accumulation of structurally unique irregular diterpenes. We discovered and described the identification and functional characterization of the relevant enzymes and their evolutionary history (Johnson et al., 2019). The work inspired application for funding from BASF, which was successfully awarded (Creativity Grant to MSU, Hamberger Lab, total direct budget $98,000). 1.4 Complementing the existing terpenoid biosynthetic toolbox with new functional parts to expand our biosynthetically accessible chemical space Expanding on our success in synthesis and purification of diterpenes using our in vitro platform of recombinant diterpene synthases, we have by October 2019 achieved production of nearly 60 diterpenes. These were all generated with the natural precursor GGDP. Intriguingly, and inspired by our earlier discovered promiscuity of the enzymes and their related pathways (e.g. Andersen-Ranberg et al., 2016, Johnson et al., 2019, Pateraki et al., 2017, Luo et al., 2016), we initiated probing of the enzymes with unnatural derivatives of GGDP. In over 150 assays, we could show that 28% of substrates were converted into novel unnatural diterpene derivatives (MSU invention disclosure in preparation, Miller, Ellsworth, Neubig and Hamberger). This preliminary data was foundation of a New Investigator grant proposal with the title "Biosynthesis of chemically diversified non-natural terpene products" for consideration under the NIH Research Grant Program (Focused Technology Research and Development) with PA number PAR-19-253. 1.5 Testing of terpenoid biosynthesis in context of modulated lipid biosynthesis in the transient Nicotiana benthamiana system and selection of optimal pathways and parts Continuing our work within Aim 4 of the GLBRC, the provisional patent application was significantly improved and filed in August 2019 (Sadre, Bibik, Benning and Hamberger; LD/Terpenoid Patent Application (3000.181WO1)). The research manuscript was accepted in Nature Communications (Sadre et al., 2019). Additional findings, specifically for the optimized pathway to the target terpenoid squalene for poplar engineering, led to the first six constructs for both plastidial and cytosolic engineering. Constructs were submitted to MSU PBROC (poplar background NM6, identified by GLBRC as target) and, in parallel, transformed into the elite poplar background P39 (in collaboration with Sean Mansfield, University of British Columbia, Canada). This allowed PhD student Jake Bibik to learn the procedure and perform the transformations.Please note that this aim also contributes to Aim 2, generation of biofuel crops. Application for year 3 funding, GLBRC was submitted (September 2019). The N. benthamiana system and the established constructs are key for a collaboration between our team and the group of Tom Sharkey (initiated September 2019) to investigate the impact of terpene biosynthesis on photosynthetic activity. 2. Adding value to the most recent generation of biofuel crops 2.1 Integrating the knowledge from 1.2 and 1.5 with engineered lines of grasses for enhanced production of lipids (in cooperation with Benning) those lines are transformed with selected terpenoid pathways to evaluate their potential application in production. Genotyping and chemotyping of identified Brachypodium lines is underway. Funding for a collaborative Research Associate was applied for to investigate Sorghum as suitable host for terpene engineering (GLBRC). Poplar lines were transformed with six constructs (MSU PBROC, UBC Dr. Mansfield) and transgenic events will be identified in Spring 2020. 2.2 Test if the entry step into the toxic phorbol esters, we have identified in paper spurge can be targeted by CRISPR and if so, generation of toxin-free lines This aim has been on hold since 2018, see previous report. 2.3 Determination of the resistance to pests or pathogens of the engineered lines (cooperating team will have to be identified) This part of the project will rely on successful identification of transgenic lines, which is currently underway (Aim 2.1). 3. Advancing our knowledge on the interaction of plants with their environment to guide future improvement of fitness and protection 3.1 Establish plant-endophyte interaction in the system Mortierella/Physcomitrella or Mortierella/Indian coleus The plant-endophyte system was fully established and has led to the identification and functional characterization of a novel fungal and plant terpene synthase, respectively (Invention disclosure 62/899,391). Transcriptomic data (provided by collaboration partner JGI) has been analyzed and indicates global reprogramming of the plant metabolism during the endosymbiotic interaction. The automated system to monitor continuously 24 cultures is established and generating data at an unexpected high resolution. Integration of the data will help assessing features of one of the most basal plant-symbiotic interactions and may shed light on evolution of symbiosis during early terrestrialization. This project was the basis for a successful funding application to participate in the 2019 Detroit Science Gallery exhibition "Depth" (Hamberger PI, co-PI Sarah Evans, Greg Bonito). 3.2 Evaluate a potential impact on plant fitness and terpenoid production The pilot experiment was successful and showed the impact of drought at unprecedented level in the moss Physcomitrella. A follow-up experiment is planned.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Invited talk, 4th International Symposium on Functional Food and Plant Metabolism, Shanghai, China, December 15-17, 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Invited talk, At the Forefront of Plant Research, Barcelona (Spain, Centre for Research in Agricultural Genomics, CRAG), May 6-8, 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Selected talk, The 14th International TerpNet Meeting, Halle (Germany, Martin-Luther-Universit�t)
August 26-30, 2019.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Sadre R, Kuo P, Chen J, Yang Y, Banerjee A, Benning C, Hamberger B. Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in leaves." Nature Communications (2019); DOI: 10.1038/s41467-019-08515-4.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Zhao D, Hamilton JP, Bhat WW, Johnson SR, Godden GT, Kinser TJ, Boachon B, Dudareva N, Soltis DE, Soltis PS, Hamberger B, Buell CR. A chromosomal-scale genome assembly of Tectona grandis reveals the importance of tandem gene duplication and enables discovery of genes in natural product biosynthetic pathways. Gigascience. 2019. doi: 10.1093/gigascience/giz005. PubMed PMID: 30698701
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Sean R. Johnson, Wajid Waheed Bhat, Radin Sadre, Garret P. Miller, Alekzander Sky Garcia, Bj�rn Hamberger. Promiscuous terpene synthases from Prunella vulgaris highlight the importance of substrate and compartment switching in terpene synthase evolution. New Phytologist. 2019; 223(1):323-335. doi: 10.1111/nph.15778.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Scientific community: international conferences, industrial meetings and invitations from national schools. Selected talk, P450s in plant diterpenoid metabolism: evolving strategies for discovery and production engineering. 14th International meeting for Cytochrome P450 Biodiversity & Biotechnology York, UK, July 15 to 19 • Guest lecture at the 9th annual Classes Without Quizzes, MSU Alumni Association, April 21, 2018 "From flavors to medicines: hijacking the treasure chest of plant natural compounds" livestream of the event: https://livestream.com/msualumni/ClassesWithoutQuizzes2018 https://livestream.com/msualumni/ClassesWithoutQuizzes2018/videos/173833886 • Meeting at MSU Technologies, BASF, May 21, 2018 contributed presentation "Terpene Synthetic Biology: from plant defense to drugs" • Invited presentation and meeting at Manus Biosynthesis, September 14, 2018 (Boston) "Plant diterpenoid metabolism: evolving strategies for discovery and production engineering" • Invited talk, The Interdisciplinary Research Forum on Getting Green Science to Market, MSU, September 21, 2018. Oral presentations by team members (1) Fani Ntana, Birgit Jensen, Hans Jørgen Lyngs Jørgensen, David Collinge and Björn Hamberger (2017) Transcriptome analysis sheds light on the Serendipita indica-tomato interaction. Int. Symposium, Microbe-assisted crop production (Austria, December 4-7) (2) F. Ntana, T. Cernava, B. Jensen, H. J. L. Jørgensen, G. Berg, D. B. Collinge and B. Hamberger (2017) Fungal endophytes - the hidden inducers of volatile terpene biosynthesis in tomato plants. Plant Biology ASPB (Hawaii, June 24-28) (3) Radin Sadre, Peiyen Kuo, Aparajita Banerjee, Christoph Benning and Bjoern Hamberger (2018) Boosting terpenoid and oil production in photosynthetic tissues of plants. Keystone Symposium on Natural Products and Synthetic Biology (California, January 21-24) (4) Sean Johnson, Sean R. Johnson, Wajid Waheed Bhat, Jacob Bibik, Aiko Turmo, Britta Hamberger, Evolutionary Mint Genomics Consortium, Bjoern Hamberger (2018) Systematic diterpene synthase discovery across Lamiaceae. 57th Annual Meeting of the Phytochemical Society of North America (Universidad Autonoma, in San Luis Potosi, Mexico, August 4-8) Press releases/public outreach Two activities need to be highlighted (i) I am mentor and hosted this year the 2018 undergraduate student team representing MSU in the international Synthetic Biology championship. Our team in 2017 won the first silver medal for MSU. (ii) After our success in 2017I co-organizedand volunteer again in the second Fascination of Plants Day at MSU. Outreach activities of the iGEM teams included lectures, demonstrations, interactive activities, public surveys at, for instance, Impression 5 Science Center, the East Lansing Public Library, the Capital Area Public Library, the Michigan Science Center and during organized North American Upper Midwest Meet-ups at MSU. MSU iGEM 2017 wins silver in Synthetic Biology World championship: https://natsci.msu.edu/news/natsci-students-on-msu-team-that-wins-silver-medal-in-synthetic-biology-competition/ http://msutoday.msu.edu/news/2017/msu-igem-team-wins-2017-silver-medal-in-boston/ 5-year NSF award to four young investigators: https://natsci.msu.edu/news/2017-10-nsf-awards-1-75-million-for-fungal-plant-research-at-msu/ 2018 Second fascination of plants day sees more than double the number of visitors: https://mps.natsci.msu.edu/news-events/news/msu-hosts-second-fascination-of-plants-day/ Interview with WKAR's Reginald Hardwick: http://wkar.org/post/msu-scientists-prepare-teach-you-anything-you-want-know-about-plants#stream/0 Synthetic biology may hold key to answering some of the most difficult science questions: https://www.canr.msu.edu/news/synthetic-biology-may-hold-key-to-answering-some-of-the-most-difficult-science-questions Changes/Problems:Change transformation system: Attempted transformation of Millet was stopped due to a lack of reproducibility.Engineering of Euphorbia lathyris was placed on hold due to technical issues regenerating transgenic events and a report by another team, describing a similar strategy in a relative species. Inspired by our succes engineering terpenoid pathways and lipid droplets in the same cell, we shifted to transformation of Camelina with seed specific promoters. Transformation was successful, lines with visible markers were recovered and terpenoid profiling on a very limited number of seeds of the first generation showed accumulation of several of the target molecules. Adjustment bioinformatics approach: After just over a year on our team, our bioinformatician postdoctoral fellow Sean Johnson accepts a permanent position in the industry. It remains to be seen if the short peroid of overlap with a graduate student provided sufficient opportunity for training. With newly hired postdoc Carlo Barnaba we hope to get critical expertise for prokaryotic expression of cytochromes P450 in our team. This forms the basis for further funding applications (follow-up SPG, collaboration with BASF, invitation to submit two prosposals). What opportunities for training and professional development has the project provided?Graduate level training: Since 2016, I have been directly involved with the Molecular Plant Sciences Graduate Program committee, and the NIH T32 Pre-doctoral Biotechnology Training Grant "Plant Biotechnology for Health & Sustainability"(NIH T32, PBHS), as Executive Committee member. Roles and activities included curriculum and course development, in particular for the two capstone courses, where speakers outside academia provide valuable insights for the students, which faculty cannot deliver. In continuation of Christoph Benning's spin on the course BMB961, I have expanded the independent development of applied biotechnological approaches. Excerpts from the recent site visit (external experts from outside MSU) and evaluation (2018): "Most impressive is the capstone class where students are encouraged over the course of a semester to develop an idea in technology transfer and work with a faculty mentor and industry mentor (as applicable) towards the commercialization.", "(...)integrative capstone course on technology transfer in the plant biotechnology arena and leadership roles in carrying out an annual symposium that attracts participants from across campus and industry.", "This course was uniformly praised by trainees and mentors alike.", "The capstone course was highly praised, it was "nothing like they had done before."". The iGEM undergraduate research experience: I and members of our team have since 2016 engaged in mentoring and training of a team of undergraduate students representing MSU at the annual Synthetic Biology World Championship (iGEM, internationally genetically engineered machine) in Boston. Academic co-mentors have been Tim Whitehead, Michaela TerAvest and Danny Ducat, and in 2018 Greg Bonito joined the mentor team. The first iGEM team at MSU in 2016 received a Bronze medal and the 2017 team brought home a Silver medal. The 2018 team which was hosted in my laboratory in the Molecular Plant Sciences Building, consists of 8 diverse students, each awarded a fellowship to cover living expenses during summer and to ensure equal opportunities. Communication of plant research to the public: We increasingly understand the critical importance of efficient communication of our research to a broad public audience. The Fascination of Plants Day is a global event under the umbrella of the European Plant Science Organization, giving exactly this opportunity. In 2017, the first year the event took place in the US, MSU spearheaded the initiative. I initiated the event and together with two postdocs, who took on the roles of event manager (Anne-Sophie Bohrer) and volunteer coordinator (Aparajita Banerjee), financial support was gathered from the Office of the Vice President for Research and Graduate Studies, the College of Natural Sciences, the MSU-DOE Plant Research Lab, the Biochemistry and Molecular Biology Program and the Department of Plant Biology. With help of a growing group of volunteers, graduate students, postdocs and faculty from six MSU units attractions were created around the theme of the event, "From Seed to Fruit", inviting the general public to explore the world of plants and algae, including fun hands-on activities for kids and adults. The 12 stations in 2017 included DNA extraction from fruits, microscope demonstrations and exploration of the world of trichomes, a terpenoid smelling station, and simulating the dispersion of fungal spores for kids while the adults had the chance to discuss topics more in depth with the researchers and learn more about the plant-related research performed at MSU. During the second Fascination of Plants Day hosted at MSU in 2018, general themes of interest were explored and attracted both more volunteers as well as approximately double the number of visitors, indicating that the event was on the right track. Oral presentations by team members (2017, 2018) (1) Radin Sadre, Peiyen Kuo, Christoph Benning and Bjoern Hamberger (2017) Boosting terpenoid and oil production in photosynthetic tissues of plants. 56th Annual Meeting of the Phytochemical Society of North America (University of Missouri, August 5-9) (2) Fani Ntana, Birgit Jensen, Hans Jørgen Lyngs Jørgensen, David Collinge and Björn Hamberger (2017) Transcriptome analysis sheds light on the Serendipita indica-tomato interaction. Int. Symposium, Microbe-assisted crop production (Austria, December 4-7) (3) F. Ntana, T. Cernava, B. Jensen, H. J. L. Jørgensen, G. Berg, D. B. Collinge and B. Hamberger (2017) Fungal endophytes - the hidden inducers of volatile terpene biosynthesis in tomato plants. Plant Biology ASPB (Hawaii, June 24-28) (4) Radin Sadre, Peiyen Kuo, Aparajita Banerjee, Christoph Benning and Bjoern Hamberger (2018) Boosting terpenoid and oil production in photosynthetic tissues of plants. Keystone Symposium on Natural Products and Synthetic Biology (California, January 21-24) (5) Sean Johnson, Sean R. Johnson, Wajid Waheed Bhat, Jacob Bibik, Aiko Turmo, Britta Hamberger, Evolutionary Mint Genomics Consortium, Bjoern Hamberger (2018) Systematic diterpene synthase discovery across Lamiaceae. 57th Annual Meeting of the Phytochemical Society of North America (Universidad Autonoma, in San Luis Potosi, Mexico, August 4-8) Training for undergraduate, international exchange graduate students and peers: Our lab is extremely popular due to the multidisciplinary nature of Plant Synthetic Biology. Every year we accept a limited number of outstanding visiting and short term visiting scholars. In 2018 we also welcomed Seitaro Mastumoto (Research Technicians, Nihon Nohyaku Co., Ltd.) andAssoc. Prof. Dr. Shagufta Perveen (May - August 2018). Thorben Hoeltkemeier, Gina Roeppischer (German Exchange, Fulbright Fellowship, May-July 2018), PhD candidates from the Marie-Curie BestPass International Training Network, 3 month research secondmends at MSU Fani Ntana (SS17), Alessandro Bergna (SS18), Eric Pereira (FS18), MSc, Jonathan Arnesen FS16, SS17 (MSc graduation August 2017). Specific undergraduate training in our team: Undergraduates Peiyen Kup and Balindile Motsa have during their training time with us picked up a lot of molecular biological skills. Both are now engaged with their own independent research projects. Undergraduates with recent co-authorship on reserach manuscripts:Alekzander Garcia (GLBRC REU), for critical metabolomic work in "Promiscuous terpene synthases from Prunella vulgaris highlight the importance of substrate and compartment switching in terpene synthase evolution" Peiyen Kuo (MSU, CE) for excellent technical assistance and performing vital experiments in the lab in "Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in plant leaves " Jonathan A. Arnesen (equal contribution, Fulbright exchange student), Daniel Moser (exchange student, University of Düsseldorf),Balindile B. Motsa (MSU, BMB) for experiments in "Engineering modular diterpene biosynthetic pathways in Physcomitrella patens" Training in scientific communication and outereach: Postdocs Aparajita Banerjee (co-organization), Sean Johnson, Wajid Waheed,graduate students Garret Miller, Davis Mathieu and undergraduates Balindile Motsa and Peiyen Kuo talk to the public during the second Fascination of Plants Day at MSU, which sees double the number of visitors comared to 2017. They run a microscope station where kids can observe trichomes, a smelling station to distinguish terpenes, DNA extraction from strawberries and "make your own DNA bracelet and DNA-model". Adults can discuss far deeper interests in the application of terpenes in the modern civilization and learn about Synthetic Biology. See details for dissemination for links to press coverage and our interview with WKAR. How have the results been disseminated to communities of interest?Target audience, the general public: Second fascination of plants day sees more than double the number of visitors: https://mps.natsci.msu.edu/news-events/news/msu-hosts-second-fascination-of-plants-day/ Interview with WKAR's Reginald Hardwick: http://wkar.org/post/msu-scientists-prepare-teach-you-anything-you-want-know-about-plants#stream/0 Synthetic biology may hold key to answering some of the most difficult science questions: https://www.canr.msu.edu/news/synthetic-biology-may-hold-key-to-answering-some-of-the-most-difficult-science-questions MSU iGEM 2017 wins silver in Synthetic Biology World championship: https://natsci.msu.edu/news/natsci-students-on-msu-team-that-wins-silver-medal-in-synthetic-biology-competition/ http://msutoday.msu.edu/news/2017/msu-igem-team-wins-2017-silver-medal-in-boston/ Target audience, scientific community: Selected talk, P450s in plant diterpenoid metabolism: evolving strategies for discovery and production engineering. 14th International meeting for Cytochrome P450 Biodiversity & Biotechnology York, UK, July 15 to 19 Conference organization/Advisor committee: International scientific advisor, The international TerpNet meeting (since 2015); Member of the Scientific Advisory Committee of the International P450 Conference for Biodiversity and Biotechnology (since 2016); Member of the organizing committee of the NIH supported 5th and 6th Annual Health and Sustainability Symposium (MSU, 2016, 2017), member of the Phytochemical Society of North America Executive Committee (since 2017). Publications and patents:Complete List of Published Work is electronically available at NCBI, MyBibliography (https://tinyurl.com/yd6h4gc7), via Google Scholar, or in the list of publications included in the files submitted: (48 articles in peer reviewed journals, over 5,700 citations; 11 patent applications, granted (8) and licensed to industrial partner: 6) Invited talk, The Interdisciplinary Research Forum on GettingGreenScience to Market, MSU, September 21, 2018. Target audience, potential industrial parters: Meeting at MSU Technologies, BASF, May 21, 2018 contributed presentation "Terpene Synthetic Biology: from plant defense to drugs" Invited presentation and meeting at Manus Biosynthesis, September 14, 2018 (Boston) "Plant diterpenoid metabolism: evolving strategies for discovery and production engineering" What do you plan to do during the next reporting period to accomplish the goals?We are on track and no immediate challenges are recognized, which could impede progress. Being awarded permanent residency in August 2018, I can now apply for NIH funding. A first exploratory meeting with the consultant Tom Hollon/Robin Buell was extremely positive. An expression of interest was invited for a full NSF proposal. An upcoming opportunity will be to further explore the inherent promiscuity of the enzymes we earlier identified with non-natural substrates, which may open the door to an entirely new level of chemical diversity. Of particular interest will be non-conventional enzymes which break evolutionary constraints and a transfer of our discovery pipeline asexpansion of our efforts from Lamiaceae to Asteraceae and Euphorbiaceae.
Impacts
What was accomplished under these goals?
1. Engineering of chassis systems for biosustainable production 1.1 Transfer of functionally characterized terpenoid biosynthetic routes to Physcomitrella Targeted metabolic engineering of the moss Physcomitrella patens, was previously achieved by integrating several pieces of foreign DNA through homologous recombination in the genome. These proof-of-principle experiments paved the way for more complex and increasingly flexible approaches for large-scale metabolic engineering. In 2017, we established stable phototrophic production of several industrially relevant terpenes and characterization of the transgenic lines was finished in 2018 (Banerjee, Arnesen, manuscript in revision). The identification of largely dispersed retrotransposon elements as suitable targets for homologous recombination has opened a new research direction currently being further explored. 1.2 Transformation of model grasses Brachypodium and Millet with pilot terpenoid biosynthetic routes Positive lines in Brachypodium for four constructs, both in the background of transgenic lines engineered for accumulation of lipid droplets and wild typewere identified and will need to be genotyped and analyzed for segregation and accumulation of terpenoids. Despite several attempts, no transgenic lines could be recovered for Millet. Since the initial publication in 2016, no other teams have reported successful transformation of Millet. This part of the project was abandoned. 1.3 Discovery of new pathways to high-value terpenoids Funding through the SPG (lead PI, Robin Buell, Pl. Biol.) and collaboration with the mint genome project allowed mapping of diterpene biosynthetic genes in 48 plant species. We built a systematic reference of chemotaxonomic and enzymatic data of diterpene metabolism. Applying this to the recently released transcriptomes of 48 mint species guided selection of 19 enzymes across 11 diverse species. Their functional characterization roughly doubled the number of scaffolds with biosynthetic access, including previously unknown, or inaccessible diterpenes. We could illustrate the approach by (i) discovery of enzymes and the related specialized diterpenes in marjoram, not known to accumulate diterpenes and (ii) the targeted discovery of the enzymes forming an agriculturally relevant scaffold in common bugle. (Provisional patent application filed August 2018; Johnson et al., manuscript under review). 1.4 (...)expanding our biosynthetically accessible chemical space Previous combinatorial assembly of neo-natural pathways to industrially relevant drug-intermediatesformed the basis for the collaborative MSU Strategic Partnership program 2017-2020 (lead PI, Rick Neubig, Pharm. & Tox.). As part of this program, our team has in 2018 biosynthesized and purified at milligram scale over 40 diterpenes, including 5 previously unknown or inaccessible compounds. These are currently being tested for anti-microbial and anti-cancer activities. Inspired by the discovery of a non-conventional diterpene synthase in Tripterygium (Hansen et al., 2017), we have adapted our strategy for identification of novel activities. Applying this targeted approach yielded the biosynthetic route of structurally extraordinary diterpenes in common selfheal (Prunella vulgaris). Our results support an emerging paradigm of substrate and compartment switching as important aspects of terpene synthase evolution and diversification. 1.5 Testing of terpenoid biosynthesis in context of modulated lipid biosynthesis (...) Our previous discovery of lipid droplets as novel intracellular storage organelle for terpenoids has inspired an engineering approach combining heterologous pathways for terpenoid formation with induction of lipid droplets. Funded earlier as collaboration with Christoph Benning through the GLBRC (2017), this approach is now integral part of the stable engineering of terpenoid pathways in biomass plant species of the newly funded GLBRC II (2018, 2019). Despite inherent advantages, such as (native) compartments and availability of reduction equivalents in form of NADPH, terpenoid biotechnology in photosynthetic tissues has remained challenging, as the engineered pathways have to compete for precursors with highly networked native pathways (and their associated regulatory mechanisms). In the present study, we established the high-yield production of terpenoids in leaves co-engineered for triacylglycerol accumulation in lipid droplets. Enhanced precursor flux and targeting of terpenoid synthesis enzymes to native and non-native compartments increased terpenoid production up to 2.6 mg terpenoids g-1 fresh weight (0.3% fresh weight). We demonstrated that the lipid droplets sequester produced terpenoids and are suitable organelles to anchor terpenoid biosynthesis steps. Our findings will have implications for future generation of stably transformed biomass crops efficiently producing industrially relevant terpenoids in photosynthetic tissues, which is of immediate relevance for the GLBRC. (Provisional patent application August 2018; Sadre et al., Manuscript in revision). As part of this process, we screened a range of enzymes catalyzing key steps in the upstream MEP pathway. The best enzymes were identified in poplar and Coleus (CfDXS, entry step of the pathway) andarchaebacteria. 2. Adding value to the most recent generation of biofuel crops 2.1 Integrating the knowledge from 1.2 and 1.5 with engineered lines of grasses for enhanced production of lipids (in cooperation with Benning) those lines are transformed with selected terpenoid pathways to evaluate their potential application in production. An panel of independent lines which were selected (see as well 1.2) will need to be genotyped and phenotyped to finish this project. 2.2 Test if the entry step into the toxic phorbol esters, (...) can be targeted by CRISPR (...) Despite early success generating callus and tissue culture from Euphorbia lathyris, no successful transformation and recovery of plants could be achieved. At the end of 2017, a team of researchers reported RNAi suppression of the diterpene synthase. While, due to this suboptimal approach only areduction of toxins could be achieved,novelty of the research is now considered limited and further efforts in this project have been placed on hold. 2.3 Determination of the resistance to pests or pathogens of the engineered lines This part of the project will rely on successful identification of transgenic lines, which is currently underway (2.1) 3. Advancing our knowledge on (...)future improvement of fitness and protection 3.1 Establish plant-endophyte interaction in the system Mortierella/Physcomitrella or Mortierella/Indian coleus Investigating the involvement of small molecules in plant fungal interactions is supported by funding through the JGI, the EU BestPass project (lead PI David Collinge, UCPH, 2015-2019) providing the scholarship of PhD student Fani Ntana, and a 5-year NSF biodimensions grant (lead PI Greg Bonito, 2017-2021). Having established growth conditions and timing for co-cultivation of one of the earliest lineages of land plants, the moss Physcomitrella patens (see also 1.1) and strains of the fungus Mortierella elongata in a pilot experiment, we submitted a large panel of samples for RNA-Seq. For medium-throughput phenotyping, we built an automated system to monitor continuously 24 cultures. Integration of the data will help assessing features of one of the most basal plant-symbiotic interactions and may shed light on evolution of symbiosis during early terrestrialization. 3.2 Evaluate a potential impact on plant fitness and terpenoid production A pilot experiment in the Mortierella/Physcomitrella interaction is currently underway to evaluate drought tolerance.
Publications
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Hamberger, B Invited presentation and meeting at Manus Biosynthesis, September 14, 2018 (Boston) Plant diterpenoid metabolism: evolving strategies for discovery and production engineering
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Hamberger, B Invited talk, The Interdisciplinary Research Forum on Getting Green Science to Market, MSU, September 21, 2018.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Dudnik A, et al. (2018)
BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits. Phytochem Rev; https://doi.org/10.1007/s11101-017-9532-2
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Luo D�, Callari R�, Hamberger B�, Wubshet SG, Nielsen MT�, Andersen-Ranberg J�, Hallstr�m BM, Cozzi F, Heider H, Lindberg M�ller B, Staerk D, Hamberger B. (2016)
Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L. Proc Natl Acad Sci U S A doi: 10.1073/pnas.1607504113. PMID: 27506796
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Jagalski V�, Barker R, Topgaard D, G�nther-Pomorski T, Hamberger B, C�rdenas M. (2016) Biophysical study of resin acid effects on phospholipid membrane structure and properties.
Biochim Biophys Acta. 2016 Aug 17;1858(11):2827-2838. doi: 10.1016/j.bbamem.2016.08.008. PMID: 27544924
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
King BC, Vavitsas K, Ikram NK, Schr�der J�, Scharff LB, Hamberger B, Jensen PE, Simonsen HT.
(2016)
In vivo assembly of DNA-fragments in the moss, Physcomitrella patens. Scientific Reports 6:25030 DOI: 10.1038/srep25030. PMID: 27126800
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Pateraki I�, Andersen-Ranberg J�, Jensen NB, Wubshet SG, Heskes AM�, Forman V, Hallstr�m B, Hamberger B�, Motawia MS, Olsen CE, Staerk D, Hansen J, M�ller BL, Hamberger B. (2017)
Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii. Elife;6. pii: e23001. doi: 10.7554/eLife.23001. PMID: 28290983
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Wubshet SG, Tahtah Y, Heskes AM, Kongstad KT, Pateraki I�, Hamberger B, M�ller BL, Staerk D (2016)
Identification of PTP1B and ?-Glucosidase Inhibitory Serrulatanes from Eremophila spp. by Combined use of Dual High-Resolution PTP1B and ?-Glucosidase Inhibition Profiling and HPLC-HRMS-SPE-NMR. J. Nat. Prod. DOI: 10.1021/acs.jnatprod.5b01128. PMID: 26960032
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Laursen T, Borch J, Knudsen C, Bavishi K, Torta F, Martens HJ, Silvestro D, Hatzakis NS, Wenk MR, Dafforn TR, Olsen CE, Motawia MS, Hamberger B, M�ller BL, Bassard JE�. (2016) Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum. Science; 354(6314):890-893. PMID: 27856908
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Andersen-Ranberg J�, Kongstad KT, Nielsen MT�, Jensen NB, Pateraki I�, Bach SS�, Hamberger B�, Zerbe P, Staerk D, Bohlmann J, M�ller BL, Hamberger B. (2016)
Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis. Angew Chem Int Ed Engl. 2016 Jan 8. doi: 10.1002/anie.201510650. PMID: 26749264
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Heskes AM�, Sundram TCM�, Boughton BA, Jensen NB, Hansen NL�, Crocoll C, Cozzi F, Rasmussen S, Hamberger B�, Hamberger B, Staerk D, M�ller BL, Pateraki I�. (2018)
Biosynthesis of bioactive diterpenoids in the medicinal plant Vitex agnus-castus. Plant J.; 93(5):943-958. doi: 10.1111/tpj.13822. PubMed PMID: 29315936
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Engineering modular diterpene biosynthetic pathways in Physcomitrella patens
Aparajita Banerjee*a,b, Jonathan A. Arnesen*a, Daniel Mosera,c, Balindile B. Motsaa, Sean R. Johnsona, and Bjoern Hamberger
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in plant leaves
Radin Sadre1,2*, Peiyen Kuo1, Jiaxing Chen1, Yang Yang1,2,3, Aparajita Banerjee1,2, Christoph Benning1,2,3,4 and Bjoern Hamberger1
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
A database-driven approach finds novel diterpene synthase activities in Lamiaceae
Sean R. Johnson1*, Wajid Waheed Bhat1,2*, Jacob Bibik1, Aiko Turmo1, Britta Hamberger1, Evolutionary Mint Genomics Consortium , Bj�rn Hamberger1
- Type:
Other
Status:
Published
Year Published:
2017
Citation:
Hamberger, B Invited talk, University of Toronto Mississauga, Graduate Program in Cell and Systems Biology, September 22
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Selected talk, P450s in plant diterpenoid metabolism: evolving strategies for discovery and production engineering. 14th International meeting for Cytochrome P450 Biodiversity & Biotechnology York, UK, July 15 to 19
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Hamberger, B " Guest lecture at the 9th annual Classes Without Quizzes, MSU Alumni Association, April 21, 2018 From flavors to medicines: hijacking the treasure chest of plant natural compounds
livestream of the event:
https://livestream.com/msualumni/ClassesWithoutQuizzes2018
https://livestream.com/msualumni/ClassesWithoutQuizzes2018/videos/173833886
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Hamberger, B Meeting at MSU Technologies, BASF, May 21, 2018 contributed presentation Terpene Synthetic Biology: from plant defense to drugs
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Scientific community: international conferences, industrial meetings and invitations from national schools. Invited talk, Graduate Program in Molecular Plant Sciences, Washington State University, Pullman, Washington, September 13, 14, 2016 Invited talk, BASF, Research Park Triangle, Raleigh, North Carolina, March 14, 2017 Invited talk, 13th International Meeting on Biosynthesis, Function and Synthetic Biology of Isoprenoids, Dalian, China, July 16 to 20, 2017 Invited talk, 56th Annual Meeting of the Phytochemical Society of North America, University of Missouri, August 5 to 9, 2017 Invited talk, University of Toronto Mississauga, Graduate Program in Cell and Systems Biology, September 22, 2017 Press releases, larger public target audience and MSU wide First MSU iGEM team https://natsci.msu.edu/news-events/news/2016-12-natsci-researchers-on-msu-team-that-medals-in-synthetic-biology-competition/ Existence of plant metabolons: https://www.eurekalert.org/pub_releases/2016-12/msu-dr122016.php http://msutoday.msu.edu/news/2016/watershed-discovery-reveals-plants-medicinal-secrets/ Public outreach Initiator and member of the team organizing the first Fascination of Plants Day at MSU (http://plantday.org/home.htm), May 20, 2017 (ASPB/EPSO). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Radin Sadre, Peiyen Kuo, Christoph Benning, Bjoern Hamberger.Boosting the production of terpenoids in lipid droplet-accumulating photosynthetic tissues. Selected talk,56th Annual Meeting of the Phytochemical Society of North America, University of Missouri, August 5 to 9, 2017 Amanda Agosto Ramos, Aparajita Banerje, Jonathan A Arnesen, Britta Hambergerand Bjoern Hamberger. High value terpenoid production in Physcomitrella patens. REU Mid-SURE Plant Genomics Symposium. Poster. Alekzander Garcia, Sean Johnson, Bjoern Hamberger. Diterpenoid Biosynthesis of the Mint family. GLBRCMid-SURE Plant Genomics Symposium. Poster. Aparajita Banerjee,Jonathan A. Arnesen, Amanda Agostoc, Britta Hambergerand Bjoern Hamberger.Engineering of Physcomitrella patens for diterpenoid production.56th Annual Meeting of the Phytochemical Society of North America, University of Missouri, August 5 to 9, 2017. Poster. Undergraduate student Davis Mathieu has been accepted into the MSU BMB graduate program, welcome to the new Spartan! Undergraduate students in the laboratory Balindile Bhekiwe Motsa (CNS Undergraduate Research Scholarship Award 2017). Effective training of graduate students in the lab through co-mentoring by Research Assistant/Postdoctoral Fellows. Member ofNIH 1T32GM110523-01 Graduate Training Grant: Plant Biotechnology for Health and Sustainability 7/2014-6/2019, Program Director Rob Last. The goal of the Plant Biotechnology for Health and Sustainability (PBHS) training program is to foster the education, training, and professional development of the next generation of interdisciplinary scientists who will assume leadership positions in biotechnology-related careers including academia, industry, government, and entrepreneurship. How have the results been disseminated to communities of interest?Communication to the public and MSU wide community: Press releases (2016): First MSU iGEM team https://natsci.msu.edu/news-events/news/2016-12-natsci-researchers-on-msu-team-that-medals-in-synthetic-biology-competition/ Existence of plant metabolons: https://www.eurekalert.org/pub_releases/2016-12/msu-dr122016.php http://msutoday.msu.edu/news/2016/watershed-discovery-reveals-plants-medicinal-secrets Initiator and member of the team organizing the first Fascination of Plants Day at MSU (http://plantday.org/home.htm), May 20, 2017 (ASPB/EPSO). For the scientific community, please see talks and publications. What do you plan to do during the next reporting period to accomplish the goals?We are on track and no immediate challenges are recognized, which could impede progress.
Impacts
What was accomplished under these goals?
Specific goals: 1. Engineering of biotechnological chassis systems for biosustainable production 1.1 Transfer of functionally characterized terpenoid biosynthetic routes to Physcomitrella Several terpenoid biosynthetic modules were introduced into the genome of a knock-out line of Physcomitrella patens, engineered for a chemical background devoid of detectable terpenoids. Tools were developed to precisely target two loci in the genome, lines were genotype-verified and production of new terpenoids demonstrated. The publication discussing our results is in preparation (Banerjee, Arnesen and co-workers). 1.2 Transformation of model grasses Brachypodium and Millet with pilot terpenoid biosynthetic routes and testing of production capacities and feasibility of the strategy Constructs for derpenoid biosynthetic pathways were generated and transformed. Lines are currently under selection for both Brachypodium (Banerjee) and Millet (Sadre). In addition we initiated transformation of Camelia using seed specific promoters (Waheed). 1.3 Discovery of new pathways to high-value terpenoids in plant species selected for their unique accumulation (in cooperation with Buell) 48 transcriptomes of the mint family were mined, complementing a comprehensive metabolite dataset of diterpenes and related molecules from several databases. We are currently focusing on (i) complementation of pathways in plants known to accumulate diterpenes, but with functionally characterized enzymes missing, (ii) de-novo discovery of diterpene synthases in species with unusual structural complexity and (iii) characterization of diterpene synthases from species lacking metabolomic data for diterpenes. A manuscript is being drafted (Johnson, Waheed). In addition several plant species are targeted where accumulation of diterpenes has been detected in trichomes. The first trichome specific RNA-Seq library has been generated, complementing those from other tissues. Discovery of terpenoid biosynthetic pathways is underway (Johnson). 1.4 Complementing the existing terpenoid biosynthetic toolbox with new functional parts to expand our biosynthetically accessible chemical space Exploring the functionalities of the diterpene synthases discovered in 1.3, we have established biosynthetic modules to four diterpene scaffolds, at full stereochemical control (Johnson, Waheed). 1.5 Testing of terpenoid biosynthesis in context of modulated lipid biosynthesis in the transient Nicotiana benthamiana system and selection of optimal pathways and parts The production was established of the terpenoid-precursors geranyl diphosphate, farnesyl diphosphate and geranylgeranyl diphosphate, and monoterpenoids (limonene), sesquiterpenoids (patchoulol, beta-caryophyllene) and diterpenoids (abietadiene) through transient expression in Nicotiana benthamiana. This included a comparison of six novel geranylgeranyl diphosphate synthases (GGDPS), key enzyme from archaebacteria, a cyanobacterium, plant and a fungus. Co-expression of diterpene synthase, a second rate limiting precursor step and plastid-targeted, optimized GGDPS, increased terpene production remarkably up to 7-fold. This study extended our current knowledge on suitable source organisms for synthetic biology. Further, we demonstrated that engineering of lipid droplets, through co-expression of WRI1, is sufficient to enhance diterpenoid production up to 3-fold. In the cytosol, sesquiterpenoid production was established through co-expression of a sesquiterpene synthase, and then boosted by co-expression of engineered HMGR and FDPS resulting in up to 10-fold higher levels. Dr. Sadre is independently coordinating this project with Dr. Aparajita Banerjee, junior associate of the GLBRC, and several undergraduate students, driving completion if this manuscript for submission to the Plant Biotechnology Journal. 2. Adding value to the most recent generation of biofuel crops 2.1 Integrating the knowledge from 1.2 and 1.5 with engineered lines of grasses for enhanced production of lipids (in cooperation with Benning) those lines are transformed with selected terpenoid pathways to evaluate their potential application in production. Pending identification of positive transgenic lines. 2.2 Test if the entry step into the toxic phorbol esters, we have identified in paper spurge can be targeted by CRISPR and if so, generation of toxin-free lines A method was developed for tissue culture of E. lathyris, prerequisite for transformation by GLBRC funded summer student Davis. He has been accepted into the graduate program at MSU. Continuation of the research is pending finding personnel. 2.3 Determination of the resistance to pests or pathogens of the engineered lines (cooperating team will have to be identified) Pending identification of positive transgenic lines. 3. Advancing our knowledge on the interaction of plants with their environment to guide future improvement of fitness and protection 3.1 Establish plant-endophyte interaction in the system Mortierella/Physcomitrella or Mortierella/Indian coleus Thesystem Mortierella/Physcomitrella was established and RNA-Seq data of a pilot study has been received. Due to the complexity of the system, work with Indian coleus is on hold until results with plant species with fully established genomes are available. 3.2 Evaluate a potential impact on plant fitness and terpenoid production Pending evaluation of the transcriptomic data, which will guide further experiments. Results obtained under the above goals provided the foundation for application for further funding. The following were awarded: MSU Foundation Strategic Partnership Grant, Lead-PI Robin Buell (MSU), co-PI Hamberger 07/01/2016-06/30/2019 Evolutionary Driven Genome Mining of Plant Biosynthetic Pathways Joint Genome Initiative supported projects Lead-PI Gregory Bonito (MSU), co-PI Hamberger 09/01/2016-08/30/2019 Functional gene network regulation in plant-fungal mutualisms in bioenergy plants and algae. MSU Foundation Strategic Partnership Grant, Lead-PI Rick Neubig (MSU), co-PI Hamberger 9/1/2017-8/31/2020 Novel plant-inspired anti-microbial and anti-cancer agents NSF DEB-1737898 Lead-PI Gregory Bonito (MSU), co-PI Hamberger 9/1/2017-8/31/2022 Phylogenetic and Functional Diversity of Tripartite Plant-Fungal-Bacterial Symbioses DOE, UW-Madison and MSU, GLBRC. co-PI Hamberger 12/01/2017
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Banerjee, A. & Hamberger, B. P450s controlling metabolic bifurcations in plant terpene specialized metabolism. Phytochem Rev (2017). https://doi.org/10.1007/s11101-017-9530-4
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Forman V, Callari R, Folly C, Heider H, Hamberger B. Production of Putative Diterpene Carboxylic Acid Intermediates of Triptolide in Yeast. Molecules. 2017 Jun 13;22(6). pii: E981. doi: 10.3390/molecules22060981. PMID: 28608823.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
. Hansen NL, Nissen JN, Hamberger B. Two residues determine the product profile of the class II diterpene synthases TPS14 and TPS21 of Tripterygium wilfordii. Phytochemistry. 2017; 138:52-56. doi: 10.1016/j.phytochem.2017.02.022. PMID: 28279524
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Hansen et al., (2016) The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily. Plant J. 2017; 89(3):429-441. doi: 10.1111/tpj.13410. PMID: 27801964
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Bjoern Hamberger, Invited talk, 13th International Meeting on Biosynthesis, Function and Synthetic Biology of Isoprenoids, Dalian, China, July 16 to 20, 2017
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Bjoern Hamberger, Invited talk, 56th Annual Meeting of the Phytochemical Society of North America, University of Missouri, August 5 to 9, 2017
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Progress 09/01/16 to 09/30/16
Outputs
Target Audience:Scientific community:international conferences. Invited talk, The 13th International Symposium on Cytochrome P450 Biodiversity and Biotechnology, Vancouver, BC, Canada from the 22th to the 26th of July 2016. Poster presentation by Aparajita Banerjee. Invited talk, The 55th Annual Meeting of the Phytochemical Society of North America, University of California, Davis, August 6 to 10, 2016. Poster presentation by Aparajita Banerjee and Britta Hamberger. Member of the Scientific Advisory Committee of the International P450 conference (Simon Fraser University, British Columbia, Canada. Member of the organizing committee for the annual Plant Biotechnology for Health and Sustainability (MSU). Students at graduate and undergraduate level: Invited talk, Graduate Program in Molecular Plant Sciences, Washington State University, Pullman, Washington, September 13, 14, 2016. Plant Genomics and GLBRC REU programs for undergraduate students, primary mentor. The first undergraduate iGEM team at MSU competing in the Synthetic Biology worldchampionship (MIT, Boston), mentor and co-supervisor. Changes/Problems:With Jiaxing Cheng joining the team for his rotation time as PhD candidate, we have the opportunity to explore engineering of Nannochloropsis, using the same functional toolbox we establish for engineering of terpenoid pathways. The current only bottleneck: We havethree MTA/CDA agreements pending at theMSU Tech Trans Office, related to materials we request from other Universities, the USDA and for collaborations where scientists wish to visit MSU for training. We are about to request other materials, which will result in an additional two docments which will need to be processed by the office. What opportunities for training and professional development has the project provided?Travel and conference participation for postdoc Aparajita Banerjee; The first international research experience for a Fulbright-funded MSc student; Undergraduate research experience in the framework of the international iGEM competition (MSU's first team competing at MIT in 2016!); Undergraduate training through summer course, REU programs GLBRC and Plant Genomics; Exposure to multidisciplinary research in areas not covered by previous experience for PhD candidate Jiaxing Cheng and Research Associates Aparajita Banerjee and Radin Sadre. PhD student Fani Ntana is currently visiting the biotech and analytics company RoomBiotic (Graz, Austria) for an industrial internship and to provide training for volatile analytics. References: 1 LuoD, Callari R,HambergerB, Wubshet SG, Nielsen MT, Andersen-Ranberg J, Hallström BM, Cozzi F, Heider H, Lindberg Møller B, Staerk D,HambergerB. (2016) Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L. Proc Natl Acad Sci U S A doi: 10.1073/pnas.1607504113. PMID: 27506796 2 King BC, Vavitsas K, Ikram NK, Schrøder J, Scharff LB,Hamberger B, Jensen PE, Simonsen HT. (2016) In vivo assembly of DNA-fragments in the moss, Physcomitrella patens. Scientific Reports 6:25030 DOI: 10.1038/srep25030. PMID: 27126800 3 Andersen-Ranberg J, Kongstad KT, Nielsen MT, Jensen NB, Pateraki I, Bach SS, Hamberger B, Zerbe P, Staerk D, Bohlmann J, Møller BL, Hamberger B. (2016) Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis. Angew Chem Int Ed Engl. 2016 Jan 8. doi: 10.1002/anie.201510650. PMID: 26749264 How have the results been disseminated to communities of interest?See previous, international conferences, workshops and graduate student programs. What do you plan to do during the next reporting period to accomplish the goals?All activities are well on track. Collaborative efforts: We are planning on one more postdoctoral hire to join the activities of engineering in collaboration with the Benning (MSU) team. MSc student Jonathan Arnesen will drive development of the Physcomitrella platform, both for engineering and thecollaboration with Gregory Bonito (MSU) (additional funding support JGI). A PhD candidate rotating student will explore, in collaboration with Eva Farré (MSU) engineering of the microalage Nannochrloropsis. Research Associate Radin Sadre will establish the metabolomic basis guiding us to select appropriate tissues within the collaborative project with Robin Buell (MSU) and Sarah O'Connor (John Innes Centre, UK) (funding support MSU foundation, strategic research program). Research Associate Aparajita Banjeree will provide constructs to enter the Brachypodium transformation pipeline (funding support GLBRC). In collaboration with Michael Feig (MSU) we are also exploring the structure function relationship of P450s (additional funding support by the JGI).
Impacts
What was accomplished under these goals?
General impact We areengineering several systems for production of plant derived metabolites of the terpenoid class. Prerequisite is knowledge of the pathways and the collection of the enzymes involved. With ourrecent publications1-3 (Luo et al., 2016, King et al., 2016, Andersen-Ranberg et al., 2016) several natural and artificial routes to commercially attractive terpenes were established and also an efficient system for transformation of the promising biotech host plant moss. For engineering of moss and biofuel crops, we have built a toolbox of functional parts, which enable production of a diverse range of diterpenes and industrially relevant terpenes of the monoterpene and sesquiterpene class. Together with collaboration partners, we are currently (i) engineering lipid droplets as interface for improved production, (ii) exploring the interaction of moss with the biotechnologically relevant fungus Mortierella and (iii) advancing our knowledge of biosynthetic pathways in medicinally relevant plants of an underexplored genus. All three projects have been awarded independent financial supportfrom the MSU foundation, the DOE through the GLBRC program and the Joint Genome Initiative, respectively. Specific details (1) 1.1 The model system Physcomitrella has been established in the lab and a propagation pipeline can generate currently 8-16 plates per week. This is the prerequisite for establishing transformation. MSc student Jonathan Arnesen (Fulbright stipend) has joined the team in August and will take on this project. Eight gene constructs related to key terpene syntheses were designed and synthesized, one terpene syntase was reveived from Prof. Joerg Bohlmann (UBC, Canada), one TPS was received from Prof. Reuben Peters (Iowa State, USA). Together these genes represent the terpenoid biosynthetic toolbox. 1.2 An alternative grass specieshas been established as a plant system and the first transformations have been performed. Material and the system is under continued development. Constructs for transformation of Brachypodium are being generated, with the major hurdle, lack of stackable selectable markers for re-transformation of previously generated lines (C. Bening Lab), being aproached. A new antibiotic may provide the replacement of non-functioning BASTA selection. 1.3 A relevant plant collection has been established, metabolomics are uderway. 1.4 Awaiting transcriptomes from 1.3 1.5 The transient transformationsystem has been set up. Initial issues with transformation efficiency, as tested by confocal laser microscopy were encountered. A new vector system, developed by G. Lomonossoff (Sainsbury Lab, UK) was requested, yet the MTA agreement has not passed MSU TTO until this date. (2) 2.1 Several target genes encoding simple, yet scientificaly relevant pathways to terpenes have been identified and all parts have been cloned from materials established at MSU and in collaboration of the MSU botanical garden, curator P.H Carrington. 2.2 A CRISPR-construct was designed, built and sequence verified, E. lathyris tissue culture was established. This project awaits new team members to proceed and might represent an ideal starting point for a rotation student. 2.3 Awaiting materials from 2.2 (3) 3.1 Mortierella/Physcomitrella interaction was successfully established. In brief, the fungal counterpart only survives in presence of the moss and it remains to be established to what degree, or wheter at all, the moss benefits from the interaction. 3.2 New team member Jonathan Arnesen is undertaking moss transformation using a novel approach: genomic targeting to specific loci in the genome.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Bj�rn Hamberger, "Discovery of terpene biosynthesis in plants: Synthetic Biology tools for production", The 55th Annual Meeting of the Phytochemical Society of North America, University of California, Davis, August 6 to 10, 2016. Invited talk.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Bj�rn Hamberger, "Its complicated: multistep pathways with multifunctional P450s in plant diterpenoid metabolism", The 13th International Symposium on Cytochrome P450 Biodiversity and Biotechnology, Vancouver, BC, Canada from the 22th to the 26th of July 2016. Invited talk.
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