| NSF Org: |
IOS Division Of Integrative Organismal Systems |
| Recipient: |
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| Initial Amendment Date: | August 21, 2013 |
| Latest Amendment Date: | July 15, 2015 |
| Award Number: | 1331173 |
| Award Instrument: | Continuing Grant |
| Program Manager: |
Irwin Forseth
IOS Division Of Integrative Organismal Systems BIO Directorate for Biological Sciences |
| Start Date: | September 1, 2013 |
| End Date: | August 31, 2017 (Estimated) |
| Total Intended Award Amount: | $535,388.00 |
| Total Awarded Amount to Date: | $535,388.00 |
| Funds Obligated to Date: |
FY 2015 = $177,182.00 |
| History of Investigator: |
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| Recipient Sponsored Research Office: |
201 OLD MAIN UNIVERSITY PARK PA US 16802-1503 (814)865-1372 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
University Park PA US 16802-7000 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | Cellular Dynamics and Function |
| Primary Program Source: |
01001516DB NSF RESEARCH & RELATED ACTIVIT |
| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.074 |
ABSTRACT
INTELLECTUAL MERIT
For optimal yields, crop plants require fixed nitrogen in the form of ammonia or nitrate fertilizers, but this requires large fossil fuel inputs and can also result in runoff which contaminates aquifers and estuaries. Unlike some microbes that have the capacity to fix atmospheric nitrogen, plants do not have this ability. The ultimate goal of this research is to engineer a novel synthetic nitrogen fixing organelle, with the long-term aim of conferring efficient nitrogen fixation in non-leguminous crop plants. However, there are significant hurdles before realizing this goal, which include high metabolic energy costs and overcoming oxygen sensitivity of the process. In this project, tools of synthetic biology will be used to engineer nitrogen fixation into a simple model system. Cyanobacteria are single-celled organisms that are evolutionarily related to plant plastids. In cyanobacteria, the engineering goals should be tractable, constituting a technological stepping stone that would lead to the engineering of nitrogen fixation into plant plastids. For this project to be successful, several objectives need to be met. First, ideal candidate gene clusters required for nitrogen fixation need to be identified. Second, using this information, tunable nitrogen-fixing gene modules, which can be precisely controlled, will be built and moved into cyanobacteria. Finally, to deal with the high metabolic energy costs of the process, a novel strategy will be employed by which extra light absorption capacity is engineered into cyanobacteria. These objectives are complex and multi-faceted, requiring tight coordination between participating laboratories. Successful completion of this research will lead to an engineered synthetic, controllable nitrogen fixing gene cluster linked energetically to light energy, which can ultimately be transferred into plastids of crop plants in the form of a 'nitroplast'.
BROADER IMPACTS
One of the major challenges of the twenty-first century is to ensure food security for the world's people. At the core of this challenge is the problem of nitrogen assimilation by non-leguminous crop plants. The goal of this project is to build a novel synthetic, controllable nitrogen-fixing module into a cyanobacterium. To achieve this goal, a team of scientific experts have been assembled from the fields of biophysics, biochemistry, molecular genetics and synthetic biology. The outcome of this research is expected to benefit basic researchers in academia as well as applied scientists through the development of new tools for cyanobacteria, and through products such as the introduction of nitrogen fixation into plants. This project is a unique opportunity for methodology exchange between U.S and U.K. scientists and for developing tools that will be freely available to the synthetic biology and cyanobacterial research communities. If successful, the work will also benefit traditional agricultural research. The preparation of the next generation of scientists is a major goal of this interdisciplinary team. The research will benefit higher education through intensive research training at the undergraduate, graduate and postdoctoral levels and through international exchange between US and UK collaborators, publications and presentations at scientific meetings. The new technologies derived from this work should provide tools and knowledge to boost nitrogen fixation capacity that could strongly impact agriculture.
PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
In non-legumous plants such as rice, high crop yields are dependent on the use of nitrogen-containing fertilizers. Were it possible to introduce nitrogen fixation into non-legumous plants, high crop yields might be possible without the use of expensive fertilizers. In nature, there is an enzyme that fixes atmospheric nitrogen into a form of ammonia but it requires energy in the form of ATP. If this energy had to be supplied by the plant, there might not be enough to sustain normal growth and metabolism as well as nitrogen fixation. The goal of the Penn State part of this project is to introduce an auxiliary photosystem into the plant so that the increased ATP requirement can be met without using any of the products of the naturally-occurring photosystems (Photosystem I and Photosystem II).
This requires the introduction of an additional photosynthetic reaction center that absorbs light in a part of the spectrum that does not interfere with visible light absorption by Photosystem I or Photosystem II. We decided to use the photosynthetic reaction center from Heliobacterium modesticaldum (Hb. modesticaldum) because it absorbs light maximally at 770 nm, which is in the near-IR part of the spectrum. Heliobacteria are anoxygenic photosynthetic bacteria that contain the simplest known photosystem, a homodimeric Type I reaction center. The heliobacterial reaction center (HbRC) consists of a homodimer of PshA, the core protein (11 transmembrane helices) containing the electron transfer cofactors, and two copies of a newly discovered PshX polypeptide (1 transmembrane helix). Its simplicity extends to the electron transfer cofactors because although it contains an interpolypeptide iron-sulfur cluster, it does not contain a quinone. The HbRC employs the pigment bacteriochlorophyll g (BChl g) that allows it to harvest near-infrared light in their native soil environment.
However, in order to assemble a functional HbRC, the biosynthesis of BChl g must first be engineered in the host organism. We decided to start by modifying the native biosynthetic pathway of BChl in Rhodobacter sphaeroides (Rba. sphaeroides) toward the production BChl g. Rba. sphaeroides is a transformable bacterium that produces bacteriochlorophyll a (BChl a), which is structurally similar to BChl g. We used a strain already modified that produces BChl b (also similar to BChl g and BChl a) and contains some modifications necessary for the production of BChl g. In order to re-direct pigment production towards BChl g in this background strain, the hydration of the C3-vinyl group of BChllide g (bacteriochlorophyll without a terpenoid tail) was blocked by deleting a gene named bchF. After culturing the strain under microoxic conditions in the dark, pigments were extracted and analyzed by HPLC and LC-MS. We observed the presence of BChl g carrying a phytyl tail (BChl gP, Figure 1), which is the tail that normally accompanies BChl a in Rba. sphaeroides. This is because the presence of the C20 molecule geranylgeranyl diphosphate (GGPP) leads to the production of an analog of BChl g with a longer tail than the authentic C15 molecule farnesyl that is attached to Bchl g (BChl gF) in Hb. modesticaldum.
Bacteriochlorphylls (BChls) are esterified with a terpenoid chain; all derive from the same C5-precursor units, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The addition of IPP units to a DMAPP result in the production of different length molecules including C15 farnesyl diphosphate (FPP) and C20 geranygeranyl diphosphate (GGPP) that will be later saturated to obtain phytyl. In order to engineer the shorter C15 tail, manipulation of the native terpenoid pathway was carried out in Rba. sphaeroides. The first step was to block the production of C20-GGPP by deleting the gene crtE. The second step was to replace the enzyme in Rba. sphaeroides that attaches the phytyl tail to the BChl g molecule with its homologue that attaches the farnesyl tail to the BChl g molecule in Hba. modesticaldum. This produced BChl g carrying a farnesyl tail with the C6=C7 and C10=C11 double bonds reduced (BChl g?2F, Figure 2). The reductions of the double bonds are carried out by the enzyme BchP, consequently in the final step we deleted the gene bchP. This finally produced BChl gF containing a C15 farnesyl tail with C6=C7 and C10=C11 double bonds (Figure 3).
These results show that the manipulation of both the native BChl and terpenoid biosynthetic pathways lead to the production of authentic BChl gF in a foreign host organism. The next step, which will be carried out under independent funding, is to express the genes pshA and pshX in Rba. sphaeroides so as to provide the protein framework for the BChl gF pigments. If this is successful, the ability of the 'synthetic' HbRC to carry out light-induced charge separation will be accomplished. If this proof-of-concept project is successful in a bacterium, the stage would be set for introduction of this photosystem into a plant.
Last Modified: 11/29/2017
Modified by: John H Golbeck
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