Progress 04/16/15 to 09/30/19
Outputs
Target Audience:Companies such as POET, South Dakota Innovation Partners (SDIP), CyanoSun Energy, and South Dakota farmers showed great interest in N2-fixing cyanobacteria as a potential source of bio-nitrogen fertilization. These companies form the base of our private sector partnerships and will provide the most direct route to commercialization. Synthetic Biology Research of N2-fixing cyanobacteria has been incorporated to three existing high level courses (MICR 450/550-Biotechnology; ABS705-Research Methodologies, MICR 438L-Molecular Biology Lab) which the PI has been teaching. The target audiences includes undergraduate students, graduate students, postdocs/visiting scientists, and higher school teachers/students as well. Changes/Problems:Major changes include: Discontinued Goal 2- Researching on regulated intramembrane proteolytic (RIP) activation of membrane-tethered transcription factors in cyanobacteria For Goal 3: We will be also working on a new project "isolation of solar-powered N2-fixing cyanobacteria from native grasslands in South Dakota". What opportunities for training and professional development has the project provided?This project served as an excellent example of integrating research and education. The knowledge and infrastructure supporting this platform project has been used in two existing courses that Dr. Zhou teaches: Micro 450/550, Applied Microbiology & Biotechnology; and ABS 705-Research Methodology. Dr. Zhou also developed a new lab-based course MICR 438L-Molecular Biology Lab in fall semester of 2014. During 2014-2019, Over 45 students and visiting scientists received hands-on research training in molecular biology and biotechnology from this grant. These included 6 PhD. students (Kangming Chen, Chuck Halfmann, Yeyan Qiu, Jaimie Gibbons, Trevor VanDenTop James Young), 7 MS graduates (Chuck Halfmann, Nathanael Braselton, Aldon Myrlie, Mathew Mckillop, Nanfang Wang, Dakota York, Maxwell Jakubiak), 18 undergraduate students, and 4 visiting scientists. How have the results been disseminated to communities of interest?The results have been disseminated mainly through peer-reviewed journal articles, scientific conferences, and invited talks. During 2014-2019 we published 27 journal articles and one book chapter, we also had at least 20 presentations in regional/ national/international conferences. In addition to these impactful publications, we were also granted one patent titled Genetically Engineered Cyanobacteria. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: (100% Accomplished) We succeeded in engineering N2-fixing cyanobacterium Anabaena sp. PCC 7120 (hereafter Anabaena) to directly convert air (N2 gas & CO2) and H2O into linalool (C10H18O), limonene (C10H16), myrcene (C10H16), farnesene (C15H24), guanidine (CH5N3) and ethylene using sunlight as the sole energy source. The 1st generation of linalool-producing Anabaena (with a plant linalool synthase gene) was confirmed to synthesize and secrete linalool. To increase linalool production, a synthetic operon coding for three rate-limiting enzymes from the MEP pathway were over-expressed in the first generation, linalool-producing Anabaena. This 2nd generation strain produced 2.8 fold more linalool than the first generation strain. Under 50 μE·m-2·s-1 illumination, the maximum linalool productivity was 35 μg/L/day/OD720 when nitrate was used as the sole nitrogen source, and 36.1 μg/L/day/OD720 when atmospheric N2 gas was the sole nitrogen source. To further increase linalool production, we created a 3rd generation of the linalool-producing Anabaena by introducing a synthetic CO2-fixing photorespiratory bypass-pathway. This synthetic pathway contained six enzymes to recover the loss of fixed carbon during photorespiration, and function as an additional O2-tolerant, CO2-fixing pathway, alongside the Calvin cycle. The 3rd generation strain showed a maximum linalool productivity of 133.7 μg/L/day/OD720, producing about 10.7-fold more linalool than the first generation strain. We also successfully transferred a plant myrcene synthase gene into cyanobacterium Anabaena. The transgenic Anabaena was confirmed to produce and secret a substantial amount of myrcene. We are currently quantifying performance of this strain. We engineered Anabaena to also produce farnesene (C15H24). A codon-optimized plant farnesene synthase gene was synthesized by IDT, and then introduced into Anabaena. Farnesene excreted from the engineered cyanobacterium volatilized into the flask head space and was recovered by adsorption in a resin column. The maximum productivity of farnesene was 69.1±1.8 μg·L−1·O.D.−1. The current sustainable energy utilization and storage technologies have been focused on carbon-rich compounds, while nitrogen-rich compounds have rarely been exploited so far. Guanidine (CH5N3) contains 71.1% N is an exemplary chemical to explore the nitrogen-based routes for energy utilization and storage. Guanidine has a variety of applications, including its use as a slow-release N fertilizer, a propellant, or as a precursor to pharmaceuticals. The conventional production of guanidine through the Frank-Caro process is fossil fuels-dependent and environmentally damaging. We engineered Anabaena to produce and secret guanidine (CH5N3) using air (N2 and CO2), mineralized water and sunlight. We transformed an ethylene-forming enzyme gene into Anabaena from Pseudomonas syringae, resulting in simultaneous production of guanidine and ethylene. The first generation strain produced guanidine at 61.5 μg L−1 D−1 using N2 gas as sole N source. We are focusing on improving its productivity by further genetic engineering. Objective 2: Molecular genetic study on cell differentiation of N2-fixing cells and oxic nitrogen-fixation in cyanobacteria (80% Accomplished) Molecular genetic study on cell differentiation: We, for the first time, succeeded in genetic transformation of A. cylindrica with pZR963 and pZR1745. PacaK, a promoter of an akinete marker gene, acaK, together with its coding region, were fused to a promoterless gfp in a pDU1-based plasmid. The resulting construct pZR963 (PacaK-acaK-gfp) was transferred into A. cylindrica by conjugation, aided by plasmid pRL2602 that contains four methylase genes to protect sequences recognized by corresponding restriction endonucleases. Fluorescence of GFP from PacaK-acaK-gfp originated principally in akinetes, with very weak fluorescence from regular vegetative cells. Fluorescence was also detected in some heterocysts and in some of what appeared to be meristematic cellular zones that contain a terminally positioned, tapered cell and its neighboring cells. Most of tapered cells divide more actively. Overexpression of acaK, through its native promoter, led to earlier, more abundant, akinete formation, sometimes distant from heterocysts. These observations suggest that overlap-expressed AcaK show a relationship between akinetes, tapering cells, and heterocysts. pZR1745 was constructed with PnifB-gfp (promoter of nifB transcriptional fusion to gfp). The transformation result revealed that expression of nifB in A. cylindrica heterocysts is regulated developmentally rather than in response to nitrogen deprivation or micro-oxic environment, providing new insights into the regulation of nif gene expression. Comparative Proteomics Study among the Heterocysts, Akinetes, and Vegetative Cells In response to environmental changes, vegetative cells of Anabaena cylindrica can differentiate into two other cell types: a heterocyst for oxic N2-fixation, and an enlarged spore called akinete for stress survival. We isolated three types of cells from A. cylindrica to identify their proteomes using LC-MS/MS. A total of 1,395 proteins were identified, including 664 proteins from akinetes, 751 proteins from heterocysts, and 1,236 proteins from vegetative cells. There were 45 proteins (33 novel proteins) found exclusive to akinetes, 57 heterocyst-specific proteins (33 novel proteins), including nif gene products, and 485 proteins exclusively in vegetative cells. Our proteomic data suggest that akinetes, unlike the typical spores of bacteria, perform unique biochemical functions that collaborate with both heterocysts and vegetative cells. The HAVe model was proposed to illustrate the metabolic network among Heterocysts, Akinetes and Vegetative cells. Interestingly, cell division proteins, DNA replication proteins, some carboxysomal proteins including RubisCO and proteins in photosystems I and II were found abundant in heterocysts, the non-dividing cells dedicated exclusively to oxic N2-fixation. The identification of the akinete and heterocyst proteomes enables us to pursue genetic study studies for a patterned differentiation of akinetes and heterocysts. Oxic nitrogen-fixation in Cyanothece sp. ATCC 51142 The unicellular cyanobacterium Cyanothece sp. ATCC 51142 was previously reported to separate the two biochemically incompatible processes of O2-producing photosynthesis and O2-labile nitrogen fixation temporally with diurnal rhythm. Here we found for the first time, a continuous-light adapted, nitrogen-deplete cultured Cyanothece sp. ATCC 51142 that exhibits strong nitrogenase activity while simultaneously carrying out oxygenic photosynthesis, raising the culture's O2 concentrations (>21%) higher than atmospheric O2 concentration). Nitrogenase activity was constitutive and constant over at least 48 h in higher O2 concentrations (>21%), and also demonstrated an absolutely dependence on light. We are currently working to quantify the rate and amount of nitrogen fixation. This is an exciting discovery! Further elucidating the adaptations underpinning this novel oxic nitrogen fixation may bring biotechnologists closer to transferring this light-dependent nitrogen fixation directly to crop leaves. Objective 3: Train undergraduate students, grad students, postdocs, visiting scientists (100% Accomplished) During this project many students, post docs, and visiting scientists received hands-on training in molecular biology and molecular genetics. These included 6 PhD. students (Kangming Chen, Chuck Halfmann, Yeyan Qiu, Jaimie Gibbons, Trevor VanDenTop James Young), 7 MS graduates (Chuck Halfmann, Nathanael Braselton, Aldon Myrlie, Mathew Mckillop, Nanfang Wang, Dakota York, Maxwell Jakubiak), 18 undergraduate students, and 4 visiting scientists.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Wang B, Dong T, Myrlie A, Gu L, Zhu H, Xiong W, Maness PC, R. Zhou, J Yu. 2019. Photosynthetic production of nitrogen-rich compound guanidine. Green Chem. 21, 29282937.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Neupane S, Schweitzer SE, Neupane A, Andersen EJ, Fennell A, R. Zhou and M P. Nepal. 2019. Identification and characterization of mitogen-activated protein kinase (MAPK) genes in sunflower (Helianthus annuus L.). Plants. 8(2):28,https://doi.org/10.3390/plants8020028.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Qiu Y, S Tian, L Gu, MB Hildreth, R Zhou. 2019. Identification of surface polysaccharides in akinetes, heterocysts and vegetative cells of Anabaena cylindrica using fluorescein labeled lectins. Arch Microbiol. 201(1):17-25.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Monono E. M, D. P Wiesenborn, J. M Vargas-Ramirez, and R Zhou. 2019. Preserving juice from industrial beets using organic acids. Transactions of the ASABE (American Society of Agricultural and Biological Engineers). 62(1):177-185.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Gibbons J, Gu L, Zhu H, Gibbons W, Zhou R. 2018. Identification of two genes required for heptadecane production in a N2-fixing cyanobacterium Anabaena sp. strain PCC 7120. AMB Express. 8(1):167.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Wang N, S Tian, L Gu, L Xu, Y Qiu, T Van Den Top, J. L Gonzalez-Hernandez, M Hildreth, S Li, R Zhou. 2018. Isolation of potential photosynthetic N2-fixing microbes from topsoil of native grasslands in South Dakota. Proceedings of the South Dakota Academy of Science. 97:117-128.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Qiu Y and Zhou R. 2019. Developmentally regulated genome editing in terminally differentiated N2-fixing heterocysts of Anabaena cylindrica ATCC 29414. 13th Workshop on Cyanobacteria. June 6-9. Boulder, CO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Trevor Van Den Top, L Gu, R Zhou. 2019. A phage-type tyrosine integrase Is responsible for excision of a nifH1 element of Anabaena cylindrica ATCC 29414. 13th Workshop on Cyanobacteria. June 6-9. Boulder, CO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Young J, M Hildreth, L Gu, R Zhou. 2019. Constitutive oxygen-tolerant nitrogenase activity In unicellular cyanobacteria. 13th Workshop on Cyanobacteria. June 6-9. Boulder, CO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Gibbons J, L Gu, R Zhou. 2019. Hydrocarbon production and biological nitrogen fixation in Anabaena sp. PCC 7120. 13th Workshop on Cyanobacteria. June 6-9. Boulder, CO.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Companies such as POET, South Dakota Innovation Partners (SDIP), CyanoSun Energy, and South Dakota farmers showed great interest in N2-fixing cyanobacteria as a potential source of bio-nitrogen fertilizer. These companies form the base of our private sector partnerships and will provide the most direct route to commercialization. Synthetic Biology Research of N2-fixing cyanobacteria has been incorporated to three existing high level courses (MICR 450/550-Biotechnology; ABS705-Research Methodologies, MICR 438L-Molecular Biology Lab) which the PI has been teaching. The target audiences includes undergraduate students, graduate students, postdocs/visiting scientists and higher school teachers/students as well. Changes/Problems:Major changes include: For goal 1: we will be also working on engineering E. coli to directly convert raw beet juices to high-value chemicals Discontinued Goal 2- Researching on regulated intramembrane proteolytic (RIP) activation of membrane-tethered transcription factors in cyanobacteria For Goal 3: We will be also working on a new project "isolation of solar-powered N2-fixing cyanobacteria from native grasslands in South Dakota". What opportunities for training and professional development has the project provided?This project also serves as an excellent example of integrating research and education. The knowledge and infrastructure supporting this platform project has been used in two existing courses that Dr. Zhou teaches: Micr 450/550, Applied Microbiology & Biotechnology; and ABS 705, Molecular Metabolic Engineering. Dr. Zhou also developed a new lab-based course MICR 438L-Molecular Biology Lab in fall semester of 2014. In 2017-2018, 12 personnel participated in this project. They included: two faculty members (Drs. Ruanbao Zhou and Liping Gu), four Ph.D. students (Jaimie Gibbons, Yeyan Qiu, Trevor VanDenTop, James Young), three MS graduates (Mathew Mckillop, Nanfang Wang, Dakota York), two undergrads (Olive Eliud, Jeffrey Koller. The students received training in biotechnology and also prepared journal articles and conference presentations. How have the results been disseminated to communities of interest?In 2017-2018 reporting period, we published 4 journal articles and we also had 7 poster presentations at national/regional conferences (see the product table). What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Engineering cyanobacteria to produce biofuels and commodity chemicals directly from CO2, water and sunlight We will continue developing a suite of engineered cyanobacteria that are separately capable of directly converting CO2 and H2O into biofuels and other commodity chemicals using free solar energy. We will also continue to engineer E. coli to directly convert raw beet juices to high-value chemicals, such as linalool, heptadecane etc. Goal 2: Researching on regulated intramembrane proteolytic (RIP) activation of membrane-tethered transcription factors in cyanobacteria We discontinued studying regulated intramembrane proteolysis (RIP) and its regulatory network in N2-fixing cyanobacteria until we get an NSF grant. Goal 3: Molecular genetic study on cell differentiation of N2-fixing cells and oxic nitrogen-fixation in cyanobacteria We will continue researching oxic N2-fixing in Anabaena sp. PCC7120, cyanothece sp.ATCC51142 etc. We will also continue to work on the heterocyst genome sequencing project which we recently started. The long-term goal of this research is to build a base for developing an oxic N2-fixing ability in all photosynthetic cells of Anabaena, eventually in plant mesophyll cells. We plan to publish at least one paper for this goal 3. We will be also working on isolation of solar-powered N2-fixing cyanobacteria from native grasslands in South Dakota for future production of bio-nitrogen fertilizer in crop fields. Goal 4: Train undergraduate students, grad students, postdocs, visiting scientists Continue to train undergraduate students, grad students, postdocs, visiting scientists as same as in this reporting period.
Impacts
What was accomplished under these goals?
Goal 1: Engineering cyanobacteria to produce biofuels and commodity chemicals directly from CO2, water and sunlight (70% Accomplished) In the past year we focused on engineering E. coli to directly convert raw beet juice to linalool. Our first genetically engineered E. coli W strain is capable of producing and secreting fragrant linalool using beet juice as sole carbon source. . The engineered E. coli W was able to produce 52.8 ± 13.4 μg linalool/L/d/optical density unit using the raw beet juice (~3% sucrose) as the sole carbon source. Directly converting raw beet juice into high-value chemicals via a genetically engineered E. coli W is innovative, and if cost-effective, it will revolutionize the beet industry across the U.S. The patentable technology gained from this research would enable the beets to become an ideal feedstock across the United States. Goal 2: Researching on regulated intramembrane proteolytic (RIP) activation of membrane-tethered transcription factors in cyanobacteria. (20% Accomplished) We discontinued studying regulated intramembrane proteolytic (RIP) activation of membrane-tethered transcription factors in cyanobacteria until we get an NSF grant to fund the work. Goal 3: Molecular genetic study on cell differentiation of N2-fixing cells and oxic nitrogen-fixation in cyanobacteria (50% Accomplished) The unicellular cyanobacterium Cyanothece sp. ATCC 51142 was previously reported to separate the two biochemically incompatible processes of O2-producing photosynthesis and O2-labile nitrogen fixation temporally with diurnal rhythm. Here we report, for the first time, a continuous-light adapted, nitrogen-deplete cultured Cyanothece sp. ATCC 51142 that exhibits strong nitrogenase activity while simultaneously carrying out oxygenic photosynthesis, raising the culture 's O2 concentrations (>21%) higher than atmospheric O2 concentration). Nitrogenase activity was constitutive, stable over many days in higher O2 concentrations (>21%), and demonstrated a dependence on light. We are currently working to quantify the rate and amount of nitrogen fixation. This is an exciting discovery! Further elucidating the adaptations underpinning this novel oxic nitrogen fixation may bring biotechnologists closer to transferring this light-dependent nitrogen fixation directly to crop leaves. Goal 4: Train undergraduate students, grad students, postdocs, visiting scientists (90% Accomplished) In the 2017-2018 reporting period, four Ph.D. students (Jaimie Gibbons, Yeyan Qiu, Trevor VanDenTop , James Young), three MS graduates (Mathew Mckillop, Nanfang Wang, Dakota York), two undergrads (Olive Eliud, Jeffrey Koller ) received hands-on training in molecular biology and molecular genetics field. Several of the students also prepared journal articles and conference presentations.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Johnson, T. J., Katuwal, S., Anderson, G. A., Gu, L., Zhou, R, Gibbons, W. R. 2018. Photobioreactor Cultivation Strategies for Microalgae and Cyanobacteria. Biotechnol Prog. 34 (4): 811-827.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Wootton, S. A., Baldwin, E. L., Gu, L., Karki, B., Halfmann, C. T., Zhou, R, Gibbons, W. R. 2017. Evaluating the efficacy of genetically engineered Escherichia coli W (ATCC9637) to produce limonene from industrial sugar beets (Beta vulgaris L.). Industrial Crops & Products. 108:248-256.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Johnson, T. J., Jahandideh, A., Isaac, I. C., Baldwin, E. L., Muthukumarappan, K., Zhou, R., Gibbons, W. R. 2017. Determining the optimal nitrogen source for large-scale cultivation of filamentous cyanobacteria. J Applied Phycology, 29(1), 1-13.
- Type:
Book Chapters
Status:
Published
Year Published:
2018
Citation:
Halfmann, C., Gu, L., Gibbons, W., Zhou, R. 2018. Synthetic Biology Enables Photosynthetic Production of Limonene from CO2 and H2O. In: Extremophilic Microbial Processing of Lignocellulosic Feedstocks to Biofuels, Value-Added Products, and Usable Power (pp. 163-189). Springer International Publishing AG, part of Springer Nature. Gewerbestrasse, Switzerland
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
McKillop, M., Gu, L., Gibbons, W., Ewumbua, M., Wiesenborn, D., Zhou, R. 2018. Engineering Escherichia coli W to produce fragrant linalool using beet juice as the sole carbon source. Annual Meeting of South Dakota Academy of Science, April 12-14, Brookings, SD.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Young, J., Hildreth, M., Zhou, R. 2018. Identification of Cell Surface Sugars in N2-fixing cyanobacterium Cyanothece ATCC 51142 Using Fluorescein Labeled Lectins. Annual Meeting of the South Dakota Academy of Science, , April 12-14, Brookings, SD.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Tian, S., Zhu, H., Gu, L., Zhou, R. 2018. N2-fixing cyanobacteria harnessed for biosolar production of nitrofertilizer. Annual Meeting of South Dakota Academy of Science, April 12-14, Brookings, SD.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Qiu, Y., Gu, L., Zhou, R. 2018. Transcriptomics of Heterocyst: Unlocking the Mystery for Solar-powered, Oxic N2- Fixation. AFRI/NIWQP/WSC Annual Project Directors Meeting, USDA-NIFA, January 29, Washington, D.C.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Wang N, J. Gibbons, L. Gu, M. Hildreth, Zhou, R. 2017. Micro-compartmentalized Subcellular Localization of a Nitrogen Fixation Protein NifV in Anabaena Heterocyst. NCB-ASM meeting, October 6-7, Green Bay, WI.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Wang N, L. Gu, L. Xu, M. Hildreth, Zhou, R. 2018. Identification of Potential Photosynthetic Nitrogen-fixing Microbes from the Topsoil of Native grasslands in South Dakota. Annual Meeting of South Dakota Academy of Science, April 12-14, Brookings, SD.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Yeyan Qiu. 2018. Proteogenomic Study of Nitrogen-Fixing Cyanobacterium Anabaena Cylindrica. Ph.D. dissertation. South Dakota State University, Brookings, SD.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Matthew McKillop. 2018. Synthetic Biology for Heterotrophic Production of High-Value Chemicals. M.S. Thesis. South Dakota State University, Brookings, SD.
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Companies such as POET, South Dakota Innovation Partners(SDIP), CyanoSun Energy, and South Dakota farmersshowed great interestin N2-fixing cyanobacteria as potential bio-nitrogen fertilizer. These companies form the base of our private sector partnerships and will provide the most direct route to commercialization. Synthetic Biology Research of N2-fixing cyanobacteria has been incorporated to three existing high level courses (MICR 450/550-Biotechnology; ABS 705-Research Methodologies, MICR 438L-Molecular Biology Lab) which the PI has been teaching.The target audiences are undergraduate students, graduate students, postdocs/visiting scientists and higher school teachers/students as well. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project also serves as an excellent example of integrating research and education. The project will improve state-of-the art in synthetic biology. The knowledge and infrastructure supporting this platform project has been used in an existing course (Micr 450/550, Applied Microbiology & Biotechnology) that Drs. Zhou and Gibbons have been teaching, and a graduate Course ABS 705-Molecular Metabolic Engineering. Dr. Zhou also developed a new lab-based course MICR 438L-Molecular Biology Lab in fall semester of 2014. In 2016-2017, 12 personnel: two faculty members (Drs. Ruanbao Zhou and Liping Gu), four Ph.D. students (Jaimie Gibbons, Yeyan Qiu, Chuck Halfmann, James Young), four MS graduates (Nathanael Braselton, Mathew Mckillop, Trevor John VanDenTop, Nanfang Wang), three undergrads (Tanner Wetzel, Trevor John VanDenTop, Olive Eliud), one postdoc (Dr. Huilan Zhu) received education training or professional development from this project. How have the results been disseminated to communities of interest?In 2017 we published 5 journal articles and we also had three poster presentations at national/international conferences. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Engineering cyanobacteria to produce biofuels and commodity chemicals directly from CO2, water and sunlight We will continue developing a suite of engineered cyanobacteria that are separately capable of directly converting CO2 and H2O into biofuels and other commodity chemicals using free solar energy. We will also shift to engineer E. coli to directly convert raw beet juices to high-value chemicals, such as linalool, myrcene, farnesene. We plan to publish at least one peer reviewed paper under this goal. Goal 2: Researching on regulated intramembrane proteolytic (RIP) activation of membrane-tethered transcription factors in cyanobacteria We are not planning to continue studying regulated intramembrane proteolysis (RIP) and its regulatory network in N2-fixing cyanobacteria until we get an NSF grant. Goal 3: Molecular genetic study on cell differentiation of N2-fixing cells and oxic nitrogen-fixation in cyanobacteria We will continue working on genetic transformation of A. cylindrica and genetically studying the cell differentiation/nitrogen fixation in A. cylindrica. Research on oxic N2-fixing will be also carried out in Anabaena sp. PCC7120, cyanothece sp.ATCC51142 etc. We will continue to work on the heterocyst genome sequencing project which we recently started. The final goal of this research is to develop an oxic N2-fixing ability in all photosynthetic cells including plant mesophyll cells. We plan to publish at least one paper for this goal 3. Goal 4: Train undergraduate students, grad students, postdocs, visiting scientistsContinue train undergraduate students, grad students, postdocs, visiting scientists as same as in this reporting period.
Impacts
What was accomplished under these goals?
Goal 1: Engineering cyanobacteria to produce biofuels and commodity chemicals directly from CO2, water and sunlight (60% complete). Cyanobacteria, like plants, have native metabolic pathways (Calvin cycle and MEP pathway) to photosynthetically convert CO2 and water into a variety of reduced carbon compounds, including GPP, the precursor for linalool. However, cyanobacteria lack the linalool synthase that plants use to convert GPP into linalool. In this year, we created three generations (1st, 2nd, and 3rd) of linalool-producing cyanobacteria. The first generation of transgenic Anabaena (with a single plant linalool synthase gene) was confirmed to continuously synthesize and secrete linalool using only air (CO2 and N2 gas), mineralized water and light energy. To increase linalool production, a synthetic operon coding for three key enzymes (DXS-IDI-GPPS) from the MEP pathway were over-expressed in the first generation of linalool-producing Anabaena . This second generation strain produced 2.8 fold more linalool than the first generation strain. Under 50 µE·m-2·s-1 illumination, the maximum linalool productivity was 35µg/L/day/OD720 when nitrate was used as the sole nitrogen source, and 36.1µg/L/day/OD720 when atmosphericN2 gas was the sole nitrogen source. To further increase linalool production, we created a third generation of the linalool-producing Anabaena by introducing a synthetic CO2-fixing photorespiratory bypass. This synthetic photorespiratory bypass route containing six enzymes is expected to avoid the loss of fixed carbon during photorespiration, and also functions as an additional O2-tolerant, CO2-fixing pathway, alongside the Calvin cycle. The third generation strain showed a maximum linalool productivity of 133.7µg/L/day/OD720 when the culture was supplemented with 1.0% CO2, producing about 10.7-fold more linalool than the first generation strain. The abstract was published in the Proceeding of International Conference on Plant Synthetic Biology and Bioengineering. Five journal papers related to goal 1 have been published along with three conference presentations. Goal 2: Researching on regulated intramembrane proteolytic (RIP) activation of membrane-tethered transcription factors in cyanobacteria (20% complete). Due to a lack of funding, we did not focus on goal 2 during the reporting period Goal 3: Molecular genetic study on cell differentiation of N2-fixing cells and oxic nitrogen-fixation in cyanobacteria (30% complete). Over-expression of acaK, through its putative promoter, led to earlier, more abundant akinete formation, sometimes distant from heterocysts. These observations suggest that overlap-expressed AcaK may show a relationship between akinetes, tapering cells, and heterocysts. This year we have been focused on knocking out avaK (a homologous gene of acaK) in Anabaena variablis-A2 to determine the role of AcaK in akinete formation. One manuscript on goal 3 is in preparation. Goal 4: Train undergraduate students, grad students, postdocs, visiting scientists (60% complete). During the reporting period, we have trained three undergraduate students, 10 grad students (6 Ph.D. students, 4 master students), one technician.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Gu L, C. Halfmann, T. VanDenTop, N. Braselton, W. Gibbons and R. Zhou (2016). Synthetic Biology of N2-Fixing Cyanobacteria for Photosynthetic Production of Perfumed Linalool from Air and Water. P20 in the Proceeding of International Conference on Plant Synthetic Biology and Bioengineering; Dec, 16-18, 2016; Miami Beach, FL.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Liping Gu, Charles Halfmann, Trevor VanDenTop, Nate Braselton, William Gibbons and Ruanbao Zhou; Poster presentation: Biosolar Synthesis of Floral Fragrance Linalool from Air and Water In Eastern South Dakota Research Symposium. May 31, 2017 Sanford Center 2301 E. 60th St. North, Sioux Falls, SD.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Yeyan Qiu, Liping Gu, Shengni Tian, Jaimie Gibbons, Jose L. Gonzalez-Hernandez, Ruanbao Zhou. Poster presentation Developmentally Regulated Heterocyst Genome Editing in Anabaena cylindrical , The 2017 Gordon Research Conference on Nucleic Acids, June 4-9, 2017; University of New England, Biddeford, ME.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Jahandideh A, T J. Johnson, N. Esmaeili, M D. Johnson, J W. Richardson, K. Muthukumarappan,
G A. Anderson, C. Halfmann, R. Zhou, WR. Gibbons (2017) Life cycle analysis of a large-scale limonene production facility utilizing filamentous N2-fixing cyanobacteria. 2017 Algal Research. 23: 1-11
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Johnson TJ, R Zhou (corresponding author)., L. Gu, JJ Gene, WR Gibbons (2016) Outlook on the potential of cyanobacteria to photosynthetically produce high-value chemicals and biofuels at an industrial scale. Bioenergetics 5:142. doi:10.4172/2167-7662.1000142
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Johnson TJ, A Jahandidehc, M D Johnsond, KAH Fieldsd, JW Richardsond, K Muthukumarappanc, Y Cao, Z Gu, C Halfmanna, R Zhou, W Gibbons (2016) Producing next-generation biofuels from filamentous cyanobacteria: An economic feasibility analysis. Algal Research. 20: 218228
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Johnson TJ, L Gu, M Hildreth, R Zhou and W R Gibbons (2016). Evaluating viable cell indicators for filamentous cyanobacteria and their application. J Microbiol Biotech Food Sci, 6(3): 883-893
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Johnson TJ, Gibbons JL, Gu L, Zhou R (corresponding author), Gibbons WR. (2016) Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review. Biotechnol Prog. doi: 10.1002/btpr.2358.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Companies such as POET, South Dakota Innovation partnerships (SDIP), CyanoSun Energy, VeraSun Energy, and KL Process Design are developing biomass to ethanol processes. These companies form the base of our private sector partnerships and will provide the most direct route to commercialization. Synthetic Biology Research of N2-fixingcyanobacteria has been incorporated to three existing high level courses (MICR450/550-Biotechnology; ABS705-Research Methodologies, MICR438L-Molecular Biology Lab) which the PI has been teaching, the target audiences are extended to undergraduate students, graduate students, postdocs/visiting scientists and higher school teachers/students as well. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project also serves as an excellent example of integrating research and education. The project will improve state-of-the-art in synthetic biology. The knowledge and infrastructure supporting this platform project has been used in an existing course (Micr 450/550, Applied Microbiology & Biotechnology) that Drs. Zhou and Gibbons have been teaching, and a graduate course ABS705-Molecular Metabolic Engineering. Dr. Zhou also developed a new lab-based course MICR438L-Molecular Biology Lab in fall semester of 2014. In 2016, 14 personnel: two faculty members (Drs. Ruanbao Zhou and Liping Gu), three Ph.D. students (Jaimie Gibbons,Yeyan Qiu, Chuck Halfmann), two MS graduates (Nathanael Braselton, Aldon Myrlie), five undergrads (ConnorBranick, MattewMckillop, Nathan Lahr, TannerWetzel, Trevor JohnVanDenTop), two postdocs (Drs. Huilan Zhu and Shengni Tian) received education training or professional development from this project. How have the results been disseminated to communities of interest?In 2016 we published 4 journal articles and we also had four poster presentations at national/international conferences. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Engineering cyanobacteria to produce biofuels and commodity chemicals directly from CO2, water and sunlight We will continue developing a suite of engineered cyanobacterial strains that are separately capable of directly converting CO2 and H2O into biofuels and other commodity chemicals using free solar energy. We plan to publishe at least one peer-reviewed paper. Goal 2: Researching on regulated intramembrane proteolytic (RIP) activation ofmembrane-tethered transcription factors in cyanobacteria We plan to systematically study regulated intramembrane proteolysis (RIP) and to further identify specific RIP protease/substrate paired systems and their regulatory network in N2-fixing cyanobacteria. We plan to publish one one peer-reviewed paper. Goal 3: Molecular genetic study on cell differentiation of N2-fixing cellsand oxic nitrogen-fixation incyanobacteria We will continue working on genetic transformation of A. cylindrica and genetically studying the cell differentiation/nitrogen fixationin A. cylindrica. Research on oxic N2-fixing will be also carried out in Anabaena sp. PCC7120, cyanothece sp. ATCC51142 etc. The final goal of this research is to develop an oxic N2-fixing ability in all photosynthetic cells including crop leaf mesophyll cells. We plan to publish at least one paper for this goal 3. Goal 4: Train undergraduate students, grad students, postdocs, visiting scientistsContinue train undergraduate students, grad students, postdocs, visiting scientists as same as in this reporting period.
Impacts
What was accomplished under these goals?
Goal 1: Engineering cyanobacteria to produce biofuels and commodity chemicals directly from CO2, water and sunlight (30% complete). The myrcene synthase gene from Norway Spruce was fused to a synthetic, dual cyanobacterial Pnir-PpsbA1 promoter and subcloned into a shuttle vector for transformation of an N2-fixing cyanobacterium Anabaena sp. PCC7120. The first generation of transgenic Anabaena (with a single plant myrcene synthase gene) was capable of photosynthetically, synthesizing and secreting myrcene, as confirmed by GC-MS. Interestingly, the transgenic Anabaena grown with atmospheric N2 gas as sole nitrogen source (N2-fixing condition) produced more myrcene than using combined nitrogen (nitrate) as nitrogen source. To further increase myrcene production, a synthetic operon coding for three key enzymes (DXS-IDI-GPPS) from the MEP pathway were over-expressed in Anabaena. This second generation strain produced at least 2-fold more myrcene than the first generation strain using atmospheric N2 gas as sole nitrogen source. These results were presented in the poster "Biosolar Synthesis of Myrcene Using CO2 and H2O via Engineered N2-fixing Cyanobacteria" at 2016 DOE JGI Genomics of Energy & Environment Meeting; March 21-24, 2016 in Walnut Creek, CA. Goal 2: Researching on regulated intramembrane proteolytic (RIP) activation of membrane-tethered transcription factors in cyanobacteria (20% complete, we didnot focus on goal 2 during the reporting peroid). Goal 3: Molecular genetic study on cell differentiation of N2-fixing cellsand oxic nitrogen-fixation incyanobacteria (20% complete). We report the first gene transfer to A. cylindrica. PacaK, a presumptive promoter of an akinete marker gene, acaK, together with its coding region, were fused to a promoterless green fluorescent protein gene (gfp) in a pDU1-based plasmid. The resulting construct (PacaK-acaK-gfp) was transferred into A. cylindrica by conjugation, aided by plasmid pRL2602 that contains four methylase genes to protect sequences recognized by corresponding restriction endonucleases. Fluorescence of green fluorescent protein (GFP) from PacaK-acaK-gfp originates principally in akinetes. Other than in very weakly expressed fluorescence from regular vegetative cells, fluorescence was also detected in some heterocysts and in some of what appear to be meristematic cellular zones that contain a terminally positioned, tapered cell and its neighboring cells, most of tapered cells divide more actively. Overexpression of acaK, through its putative promoter, led to earlier, more abundant, akinete formation, sometimes distant from heterocysts. These observations suggest that overlap-expressed AcaK may show a relationship between akinetes, tapering cells, and heterocysts. One manuscript on goal 3 is in preparation. Goal 4:Train undergraduate students, grad students, postdocs, visiting scientists (20% complete). During the reporting peroid, we have trained five undergraduate students, 5 grad studnets, one visiting scientist, one technician.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Chen K, Zhu H, Gu L, Tian S and R Zhou (2016) Target gene inactivation in Anabaena sp. PCC 7120. Bio-protocol, 6:15, Aug 5, 2016 (published online).
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Johnson TJ, C. Halfmann, JD. Zahler, R Zhou, W Gibbons (2016). Increasing the tolerance of filamentous cyanobacteria to next-generation biofuels via directed evolution. Algal Research, 18: 250256
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Johnson TJ, A Jahandideh, IC. Isaac, E L Baldwin, K Muthukumarappan, R Zhou, W Gibbons (2016). Determining the optimal nitrogen source for large-scale cultivation of filamentous cyanobacteria. J Appl Phycol (2016). doi:10.1007/s10811-016-0923-3
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Johnson TJ, Zahler JD, Baldwin EL, Zhou R, Gibbons WR. (2016). Optimizing cyanobacteria growth conditions in a sealed environment to enable chemical inhibition tests with volatile chemicals. J Microbiol Methods. 126:54-9.
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Progress 04/16/15 to 09/30/15
Outputs
Target Audience:Companies such as POET, South Dakota Innovation partnerships (SDIP), CyanoSun Energy, VeraSun Energy, and KL Process Design are developing biomass to ethanol processes. These companies form the base of our private sector partnerships and will provide the most direct route to commercialization. Metabolic engineering cyanobacteria has been incorporated to three existing high level courses (MICR450/550-Biotechnology; ABS705-Research Methodologies and Micr438-Molecular Biology Lab)which the PI has been teaching, the target audiences are beingextended to undergraduate students, graduate students, postdocs/visiting scientists and higher school teachers/students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In 2015, the project provided hands-on training for five undergraduate students, two postdocs/visiting scientists, five graduate students and one technician in my laboratory. How have the results been disseminated to communities of interest? The PI Dr. Zhou presented the research findings at the NC-Sun Grant annual Meetings (03/2015)"Engineering N2-fixing cyanobacteria to synthesize myrcene (C10H16) from air, water and sunlight" Gu L, Halfmann C, Xiang X, Gibbons W. R. Zhou (2015) Engineering N2-fixing cyanobacteria to synthesize perfumes from air, water and sunlight. P112 in The 10th Annual DOE Joint Genome Institute Genomics of Energy & Environment. Meeting March 24 - 26, 2015 in Walnut Creek, California. R Zhou (2015) "Synthetic Biology for Direct Production of Jet Fuel and High-value Chemicals from Air and Water", 1st International Conference on Science at the Sanford Underground Research Facility, Rapid city, SD, 05/17 2015 -05/2020/2015. Braselton N, Zhou R (2015) Engineering N2-fixing cyanobacteria to synthesize fuel ethanol from air, water and sunlight. 1st International Conference on Science at the Sanford Underground Research Facility, Rapid city, SD, 05/17 2015 -05/2020/2015. The four papers on research findings were published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?1. Continue engineering cyanobacteria to produce biofuels and commodity chemicals directly from CO2, water and sunlight 2. Researching on regulated intramembrane proteolysis (RIP) in cyanobacteria The long-term goal of this project is to systematically study regulated intramembrane proteolysis (RIP) and to further identify specific RIP protease/substrate paired systems and their regulatory network in N2-fixing cyanobacteria. 3. Molecular genetic study on cell differentiation of N2-fixing cellsand oxic nitrogen-fixation incyanobacteria There has been little or no genetic study on the differentiation of akinetes and heterocysts in A. cylindrica due to the lack of a transformation approach. This project is to develop a genetic transformation approach to A. cylindrica. This approach will enable us to be a pioneer in genetically studying the cell differentiation/nitrogen fixationin A. cylindrica. Researching on oxic N2-fixing will be also carried out in Anabaena sp. PCC7120, cyanothece sp. ATCC51142 etc. The final goal of this research is to develop an oxic N2-fixing ability in all photosynthetic cells including crop leaf mesophyll cells. 4. Train undergraduate students, grad students, postdocs, visiting scientists
Impacts
What was accomplished under these goals?
1) We had succeeded in engineering N2-fixing cyanobacteria to directly convert air (N2 gas & CO2) and H2O into linalool (C10H18O), potential drop-in fuel, using sunlight as sole energy. The engineered cyanobacterial strain serves as a cellular factory to be capable of both synthesis and secretion of linalool, placing linalool in a unique position ideal for potential commercial applications. (2) We had successfully transferred a plant MyrS gene constructed in pZR966 into cyanobacterium Anabaena sp. PCC7120. The plant myrcene synthase was confirmed to be expressed by Western blot.The first generation of transgenic cyanobacteria has been confirmed to produce and secret a significant amount of myrcene. (3) We have succeeded in engineering Anabaena sp. PCC7120 to produce farnesene (C15H24) using air, mineralized water and sunlight. The work was recently published in Applied Microbiology and Biotechnology. (4) We, for the first time, succeeded in genetic transformation of A. cylindrica. With an aid of pRL2602, we successfully transformed three pDU1-based cargo plasmids into A. cylindrica. The transformation results reveal (i) the overlapping expression of PacaK and PnifB in akinetes and in heterocysts; (ii) that nifB in A. cylindrica is regulated developmentally rather than in response to nitrogen deprivation, providing new insight into regulation of nif gene expression.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Johnson TJ, Hildreth MB, Gu L, Zhou R, Gibbons WR (2015). Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria. J Microbiol Methods. 113:57-64.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
R. Zhou, L. Gu, W. Gibbons and C. Halfmann (2015). On the Cyanofactory Floor: Next-generation biofuel. International Innovation, A renewable future V178:118-119.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Chen K, X. Xu, L. Gu, and R. Zhou (2015). Simultaneous Gene Inactivation and Promoter Reporting in Cyanobacteria. Appl Microbiol Biotechnol. 99(4):1779-93
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Xu X, L Gu, P He, and R Zhou (2015). Characterization of five putative aspartate aminotransferase genes in the N2-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120. Microbiology. 161(6):1219-30
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