ENGINEERING SOLAR-POWERED N2-FIXING CYANOBACTERIA FOR AGRICULTURAL AND INDUSTRIAL APPLICATIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1019554
• Project Start Date: Oct 1, 2019
• Project End Date: Sep 30, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007

Performing Department
Biology & Microbiology

Non Technical Summary
Cyanobacteria, with an evolutionary history of more than 2.5-billion years, are arguably the most successful group of life forms on Earth. They can be found in almost every terrestrial and aquatic habitat. Cyanobacteria have been used as a model organism to study many fundamental biological processes, including photosynthesis, nitrogen (N2) fixation, cell differentiation and development, stress response, and bioenergy production. Many cyanobacteria are capable of simultaneously carrying out agriculturally important O2-producing photosynthetic CO2-fixation and O2-labile N2-fixation, the two fundamental pathways that all life forms rely upon.Given their unique capabilities and their genetic tractability, cyanobacteria are one of the best organisms to engineer to solve agricultural and bioenergy related problems. This project will seek to accomplish the following objectives:Objective 1. To understand the molecular genetics and proteogenomics of cell differentiation in N2-fixing cyanobacteriaObjective 2. To develop a cellular "cyanofactory" platform that is able to directly convert air (CO2 & N2) and water into high-value chemicals using sunlightObjective 3. To isolate and characterize solar-powered N2-fixing cyanobacteria from native ecosystems and develop methods to integrate them into agricultural systems to replace synthetic fertilizersTo accomplish objective 1 we will first identify specific proteins in distinct cell types, then inactivate the specific genes coding for these proteins, and look for any resulting changes in cell differentiation in N2-fixing cyanobacteria, such as heterocyst/akinete differentiation. An alternative approach will be to use transposon mutagenesis to generate mutants that fail to form akinetes or heterocysts, then to identify the novel genes required for cell differentiation through inverse PCR and DNA sequencing approaches.To accomplish objective 2 we will use a synthetic biology approach to develop a cellular cyanofactory, the genetically engineered cyanobacteria, to directly convert air (CO2 & N2) and water into fuel molecules (e.g. limonene) and high-value chemicals using sunlight.To accomplish objective 3 we will isolate solar-powered N2-fixing cyanobacteria from native ecosystems across South Dakota and then validate their nitrogen fixation capability. We expect to isolate 2-3 strains with high nitrogen-fixation ability, and then test in greenhouse trials to replace synthetic fertilizers. The long-term goal is to develop a method to integrate the best strains into agricultural systems to enable these isolated N2-fixing cyanobacteria to serve as "solar-powered living, sustainable N-fertilizer factories" to reduce or replace synthetic fertilizers and also improving soil health for crop fields.To this point, development of sustainable economy has primarily focused on the carbon-based economy, while the nitrogen-based economy has had relatively little attention. However, dinitrogen (N2 gas) is far more abundant (80%) than CO2 (0.04%) in atmosphere. This project will focus on the nitrogen-based economy using N2-fixing cyanobacteria for agricultural and industrial applications. N2-fixing cyanobacteria are a unique group of organisms that are capable of carrying out both photosynthetic CO2-fixation and solar-powered N2-fixation, the two fundamental pathways that all life forms rely upon. To understand how they do so would be of huge benefit to society. These benefits would include:Genetically engineered N2-fixing cyanobacteria that can produce many nitrogen-rich chemicals, biofuels, all kinds of industrial chemicals that will gradually replace petroleum-based products with bio-solar products.Enable N2-fixing cyanobacteria in crop fields to serve as in situ "solar-powered living, sustainable N-fertilizer factories" to reduce or replace synthetic N-fertilizers and also improve soil health. In addition to reduced costs, cyanobacteria-based nitrogen fixation using solar energy and CO2 would also alleviate the two global problems: fossil fuel depletion and environment degradation due to elevated CO2 emissions.

Animal Health Component

10%

Research Effort Categories

Basic

70%

Applied

10%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	0199	1040	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
0199 - Soil and land, general;

Field Of Science
1040 - Molecular biology;

Keywords

biofuel & bioenergy

green chemicals

solar-powered nitrogen fixation

n2-fixing cyanobacteria

cell differentiation

bio-nitrogen fertilizer

isoprene

rubber

hydrocarbons

terpenes

Goals / Objectives
The overarching goal of this project is to engineer cyanobacteria to produce agricultural and industrial products in a more economical and sustainable manner than is presently possible. The project has three specific objectives:Objective 1. To understand the molecular genetics and proteogenomics of cell differentiation in N2-fixing cyanobacteriaObjective 2. To develop a cellular "cyanofactory" platform that is able to directly convert air (CO2 & N2) and water into high-value chemicals using sunlightObjective 3. To isolate and characterize solar-powered N2-fixing cyanobacteria from native ecosystems and develop methods to integrate them into agricultural systems to replace synthetic fertilizers

Project Methods
Objective 1. To understand the molecular genetics and proteogenomics of cell differentiation in N2-fixing cyanobacteriaVegetative cells (V) of A. cylindrica can differentiate into two other cell types: a heterocyst (H) for oxic N2-fixation, or an enlarged spore called akinete (A) for stress survival. Heterocysts inhibit nearby cells from differentiating into heterocysts but can induce nearby cells to become akinetes, a rare embryogenetic induction in prokaryotes. The primary goal of objective is to identify novel genes that control the differentiation process for akinetes and heterocysts. We will use molecular genetics, genomic, transcriptomic, and proteomics approaches to accomplish this objective. Single or double cross-over recombination will be used to inactivate specific protein coding genes to isolate mutants defective in akinete/heterocyst formation. We have previously identified these candidate genes from a comparative proteomics study and transcriptomics data. An alternative approach will be to use transposon mutagenesis to generate mutants that fail to form akinetes/heterocysts. We will then use inverse PCR combined genomic DNA sequencing to identify the genes mutated by transposon Finally, we will put the wildtype gene back to the transposon-generated mutants (complementation) to confirm that the mutated genes are responsible for the phenotype.Objective 2. To develop a cellular "cyanofactory" platform that is able to directly convert air (CO2 & N2) and water into high-value chemicals using sunlightModern molecular biology methods will be used to genetically engineer cyanobacteria to produce biofuels and commodity chemicals directly from air (CO2 & N2), water and sunlight. We already succeeded in genetically engineering N2-fixing cyanobacteria to produce limonene (C10H16), linalool (C10H18O), and farnesene (C15H24) at low rates. In this objective we will seek to improve productivity, using isoprene as a model product. We anticipate completing the following steps:Step 1: Engineer N2-fixing cyanobacteria to produce isoprene using air (CO2 & N2) and H2O by introducing a plant isoprene synthase gene.Step 2: Introduce an optimized exogenous mevalonate pathway (MVA pathway) into the isoprene-producing strain to further improve isoprene production.Step 3: Block or reduce carbon flow to competing pathways to maximize production and excretion of isoprene. The major storage carbohydrate in Anabaena is glycogen. We will block glycogen synthesis by inactivating glucose-1-phosphate adenylyltransferase, which catalyze the first committed step in glycogen biosynthesis. Only one glucose-1-phosphate adenylyltransferase gene was found in cyanobacterial genomes. We will block glycogen synthesis, for instance, in Anabaena by inactivate All4645 gene using either a single or double cross-over knockout approach that was developed by the PI.Objective 3. To isolate and characterize solar-powered N2-fixing cyanobacteria from native ecosystems and develop methods to integrate them into agricultural systems to replace synthetic fertilizersTo isolate N2-fixing cyanobacteria we will collect approximately 300 topsoil samples from native ecosystems, no-tilled crop fields, and tilled crop fields across South Dakota and neighboring states. Samples will be inoculated into N2-free growth media to isolate N2-fixing cyanobacteria. Isolates will be repeatedly subcultured onto a solid growth medium to ensure purity. Isolates will then be identified via genomic techniques.The N2-fixing capability of the isolates will be assessed by conducting acetylene reduction assays to identify strains with the most active nitrogenase enzymes. We will down-select the most productive of these isolates in terms of growth and N2-fixation rates. We will then conduct genome sequencing to identify and characterize novel N2-fixing (nif) genes.To assess the potential benefits of these cyanobacteria on plant growth we will first grow seedling plants in an agar-based, nitrogen-free growth medium, either with or without the cyanobacteria isolates. Enhanced plant growth in the presence of N2-fixing cyanobacteria will supportive of the hypothesis that that the cyanobacteria can contribute fixed nitrogen to the plant. Cyanobacteria showing positive effects on plant growth will then be tested in greenhouse trials, first in sterile soil and then in actual field soil. We will evaluate different application rates and assess cyanobacteria growth, nitrogen fixation, and plant responses. Trials may also be repeated in the same soil over several years (with and without inoculation) to assess the persistence of the N2-fixing cyanobacteria. From this information we should be able to determine how various application rates will benefit N availability and crop production.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Companies such as POET, South Dakota Innovation Partners (SDIP), CyanoSun Energy, and South Dakota farmers showed great interest in use of N2-fixing cyanobacteria for production of nitrogen-rich compounds such biofuels, biochemicals, 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 and farmers. Changes/Problems: Nothing Reported 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 three existing courses that Dr. Zhou teaches: Micro 450/550, Applied Microbiology & Biotechnology; and ABS 705-Research Methodology, MICR 438L-Molecular Biology Lab. During 2019-2020, total 10 personnel received hands-on research training in molecular biology and biotechnology from this grant. These included three PhD students: Jaimie Gibbons, Trevor VanDenTop, James Young, two MS graduates: Dakota York, Maxwell Jakubiak and Jianjun Liu (visiting graduate student), three undergraduate students: Nathan Lahr, Tanner Gauer and Mohammad Al Radhwan. One post doc: Dr. Jaimie Gibbons. 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. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. To understand the molecular genetics and proteogenomics of cell differentiation in N2-fixing cyanobacteria We continue study the heterocyst/akinete differentiation using genetic approach combined with genomics, proteomics, and transcriptomics. We will also use RNAseq technology to obtain heterocyst transcriptome and using Nanopore sequencing technology to obtain the complete genomic sequence of heterocyst. We may also start knocking out the heterocyst/akinete-specific genes. Objective 2. To develop a cellular "cyanofactory" platform that is able to directly convert air (CO2 & N2) and water into high-value chemicals using sunlight We will be focusing on improving guanidine's productivity by further genetic engineering of the fastest-growing, heterocyst-forming cyanobacterial strain ATCC 33047. Alternatively, we will also start engineering of the unicellular N2-fixing cyanobacterium, Cyanothece sp. ATCC 51142, for production of nitrogen-rich compounds (ammonia & guanidine, essential amino acids, isoprene et al.) using air (CO2 & N2) and sunlight. Objective 3. To isolate and characterize solar-powered N2-fixing cyanobacteria from native ecosystems and develop methods to integrate them into agricultural systems to replace synthetic fertilizers We will continue to isolate, purify, characterize the potential N2-fixing cyanobacterial strains, and validate their nitrogen fixation ability in next year. We expect to obtain 2-5 powerful N2-fixing cyanobacterial strains for practical application in agriculture and industry.

Impacts
What was accomplished under these goals? Objective 1: To understand the molecular genetics and proteogenomics of cell differentiation in N2-fixing cyanobacteria (30% Accomplished) 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. We found 45 proteins (33 novel proteins) exclusively 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 physiological 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, 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 (e.g. specific genes knockout) for a patterned differentiation of akinetes and heterocysts in A. cylindrica. This work "Unique Proteomes Implicate Functional Specialization across Heterocysts, Akinetes, and Vegetative Cells in Anabaena cylindrica" was recently published as a preprint in bioRXiv. Objective 2. To develop a cellular "cyanofactory" platform that is able to directly convert air (CO2 & N2) and water into high-value chemicals using sunlight. (20% Accomplished) 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 fuel-dependent and environmentally damaging. We successfully engineered Anabaena sp. PCC7120 (a heterocyst-forming filamentous cyanobacterium) to produce and secret guanidine (CH5N3) using air (N2 and CO2), mineralized water, and sunlight. The first generation strain produced guanidine at 61.5 μg L−1 D−1 using N2 gas as sole N source. This work "Photosynthetic production of nitrogen-rich compound guanidine" was published in Green Chemistry (2019). We are now focusing on improving its productivity by further genetic engineering. Objective 3. To isolate and characterize solar-powered N2-fixing cyanobacteria from native ecosystems and develop methods to integrate them into agricultural systems to replace synthetic fertilizers (20% Accomplished) N2-fixing cyanobacteria have been playing critical roles in maintenance of soil fertility and soil health that harmonize the soil biological, chemical, and physical properties to sustain huge annual biomass production (no need of N-fertilizer) in native ecosystems. This project is to survey and isolate N2-fixing cyanobacteria in South Dakota natural ecosystems (native grasslands/forests/Badlands), so that we can return these "bugs" to crop fields where the "bugs" may be extinct due to heavily applied chemical N-fertilizer and frequent tillage. The findings from this project will help develop N2-fixing cyanobacteria as a producer of N-fertilizer in crop field, and may provide a practical application in non-till cropping systems. The long-term goal of this project is to enable these isolated N2-fixing bugs in crop fields as in situ "solar-powered living N-fertilizer factories" to reduce current cost for N-fertilizer and also improving self-sustainable soil health in agricultural lands. We have isolated 15 potential N2-fixing cyanobacterial strains from the 244 topsoil samples collected in natural ecosystems or non-till cropping systems. We will continue to purify, characterize these 15 potential N2-fixing cyanobacterial strains, and validate their nitrogen fixation ability in next year.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang B, Dong T, Myrlie A, Gu L, Zhu H, Xiong W, Maness PC, Zhou R, Yu J. 2019. Photosynthetic production of nitrogen-rich compound guanidine. Green Chem. 21, 29282937.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Sobhana A, Muthukumarappana K, Wei L, VanDenTop T, Zhou R. 2020. Development of an activated carbon-based nanocomposite film with antibacterial property for smart food packaging. Materials Today Communications. https://doi.org/10.1016/j.mtcomm.2020.101124.
• Type: Book Chapters Status: Accepted Year Published: 2020 Citation: Young J, Gu L, Gibbons W, Zhou R. 2020. Harnessing solar-powered N2-fixing cyanobacteria for the bioNitrogen economy. In Cyanobacteria: metabolic engineering and biotechnology, ed: Paul Hudson, Publisher: Wiley.
• Type: Journal Articles Status: Other Year Published: 2020 Citation: Qiu, Y, Gu L, Br�zel V, Whitten D, Hildreth M, Zhou R. 2020. Unique proteomes implicate functional specialization across heterocysts, akinetes, and vegetative cells in Anabaena cylindrica. bioRXiv. doi: https://doi.org/10.1101/2020.06.29.176149