GENETIC ENGINEERING OF CYANOBACTERIA TO PRODUCE HIGH-VALUE PROTEINS USING ATMOSPHERIC N2 GAS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1030536
• Grant No.: 2023-67022-39594
• Cumulative Award Amt.: $300,000.00
• Proposal No.: 2022-10766
• Project Start Date: Apr 1, 2023
• Project End Date: Mar 31, 2026
• Grant Year: 2023
• Program Code: [A1531]- Biorefining and Biomanufacturing

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

Performing Department
(N/A)

Non Technical Summary
Proteins are essential nutrients for humans/animals. There are nine essential amino acids (EAA) which humans must obtain from their diets to prevent protein malnutrition. Most foods contain nine EAA, but they are NOT in sufficient quality and quantity required for protein nutrition. Thus, humans have to consume excessive proteins from various food sources to meet the EAA requirement. Although EAA deficiency may be overcome, over-consumption of proteins may risk developing health problems such as kidney stones, obesity, cardiovascular disease, and cancers.As the world's population increases rapidly and food resources are limited, it is more pressing than ever to seek alternative, sustainable production of quality proteins.This seed project will use atmospheric N2 gas as a sole nitrogen source to produce a specially designed, quality nutritional protein that is not found in nature. To our best knowledge, this is pioneering research on production of all nine essential amino acids (EAA) simultaneously, by introducing a single synthetic gene coding for EAA-rich Protein (EarP) into a solar-powered N2-fixing cyanobacteria. Certainly, this novel approach with the EarP gene we creat will be patentable. If successful, we can create a "microbial protein factory" to produce any designed, high-value, synthetic proteins for many other applications. Eventually, we may transfer this EarP gene to edible blue-green algae, vegetables, crops and milk-cow to produce further optimized EarP for improving human/animal protein nutrition.

Animal Health Component

20%

Research Effort Categories

Basic

60%

Applied

20%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
701	4010	1040	80%
502	4010	1010	10%
502	4010	1000	10%

Knowledge Area
701 - Nutrient Composition of Food; 502 - New and Improved Food Products;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1040 - Molecular biology; 1010 - Nutrition and metabolism; 1000 - Biochemistry and biophysics;

Keywords

n2-fixing cyanobacteria

genetic engineering

high-value protein

Goals / Objectives
The long-term goal of this project is to genetically engineer N2-fixing cyanobacteria to be a commercially viable "cyanofactory" to produce high-value N-rich and/or C-rich bioproducts using atmospheric N2 gas as a sole nitrogen source. This seed project will focus on genetically engineering Anabaena sp. PCC 7120 (hereafter Anabaena) to produce and secrete a designed optimized proportional EAA-rich Protein for quality human/animal protein nutrition, using air (CO2 & N2), mineralized H2O, and sunlight.Objective 1: Design and synthesize the EarP gene.Objective 2: Construct expression plasmids for EarP over-production and secretion.Objective 3: Production, purification, and verification of EarP.Objective 4: Inactivate a cyanophycin synthetase gene to shunt Anabaena's N-flux from producing cyanophycin to the production of EarP.

Project Methods
Objective 1: Design and synthesize the EarP gene.The EarP amino acid sequence was designed based on: a) the optimized EAA mole ratio based on the recommended EAA composition profile, and b) the optimized di-peptide bonds based on their frequency in Anabaena's predicted proteome. The resulting EarP amino acid sequence was used to generate the codon optimized nucleotide sequence according to Anabaena's codon usage table. The EarP's optimized EAA mole ratio was derived from a quality nutritional protein recommended by the Institute of Medicine's Food and Nutrition Board and from EAA composition of whole body proteins from six mammalian species.Objective 2: Construct expression plasmids for EarP over-production and secretion.Standard molecular biology techniques/methods and molecular genetics approaches to genetically engineer N2-fixing cyanobacteria will be used to produce novel quality nutritional proteins. To over-express and purify the EarP protein, the different synthetic versions of EarP gene, the 2x earP codon optimized gene with an optimized translation initiation sequence (ribosome binding sequence), a six-histidine tag (H6), secretory signal peptide sequences (SPN-alr4550, SPN-alr0608, SPN-all4499 and SPalr0267-C80) will be fused to the proper positions of 2xearP and be synthesized at IDT. The synthesized gene fragments will be subcloned into the pZR1188 expression vector. The six-histidine (H6) fused to EarP can be used for both Western blot detection and Ni-NTA affinity purification of the H6-tagged EarP from the culture media and/or cell lysates. To maximize the EarP production, a dual Anabaena promoter PpsbA1-Palr0267 will be designed to drive the EarP gene expression.Objective 3: Production, purification, and verification of EarP.Various versions of 2xearP containing plasmids (Objective 2) will be transformed into Anabaena sp. PCC7120 by conjugation. The Anabaena strains bearing various versions of pEarP plasmids will be grown in a 500 ml flask supplied with BG110 medium (mineralized water without combined nitrogen). To determine which fraction contains the majority of EarP proteins, total proteins from the culture media, whole cell lysate, and 12000 x g supernatant and the pellet, respectively, will be analyzed via SDS-PAGE followed by Western blot with anti-His antibodies. Then, Ni-NTA affinity resin will be used to purify the His-tagged EarP proteins by Ni-NTA affinity chromatography. Further quantification and verification of 2xEarP can be achieved by Sanger DNA sequencing, Western Blot or MALDI-TOF mass spectrometry.Objective 4: Inactivate a cyanophycin synthetase gene to shunt Anabaena's N-flux from producing cyanophycin to the production of EarP.The gene all3879 encoding for cyanophycin synthetase in Anabaena sp. PCC7120 will be inactivated through a single-crossover approach described by Jamie Gibbons et al. (2022). The cargo plasmid containing all3879 internal fragment will be transformed into Anabaena by conjugation to inactivate all3879 to produce SR3879 mutant strain, then, the highest EarP production/secretion construction will be transformed into SR3879. Finally, the EarP yield will be quantified using the methods described in Objective 3.

Progress 04/01/24 to 03/31/25

Outputs
Target Audience:Companies involved in Biosynthesis and Bioproduction such as Houdek, Dakota Bioworx, Medgene, POET, South Dakota Innovation Partners (SDIP), and animal feed companies. These companies form the basis of our private sector partnerships and will provide the most direct route to commercialization. Genetic engineering of N2-fixing cyanobacteria has been incorporated into two existing high-level courses (MICR 450/550-Biotechnology; MICR 438L-Molecular Biology Lab) which the PI has been teaching. The target audiences include undergraduate students, graduate students, postdocs/visiting scientists, and high school teachers/students as well as 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 two existing courses that PI/Co-PI teaches: Micro 450/550, Applied Microbiology and Biotechnology; and MICR 438L-Molecular Biology Lab. There were 15 students enrolled in MICR450/550; and 9 students in MICR438L during the report period. During 2024-2025, a total of 5 personnel received hands-on research training in molecular biology and biotechnology from this grant. These included three PhD students: Taufiq Nawaz, Dillon Nelson, Cayden Budd and one MS student Maxwell Jakubiak as well as one visiting scientist Dr. Shah Fahad. 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. One review paper titled "Sustainable protein production through genetic engineering of cyanobacteria and use of atmospheric N2 gas" has been published in Food and Energy Security, What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Design and synthesize the EarP gene. 100% Accomplished; N/A Objective 2: Construct expression plasmids for EarP over-production and secretion. 65% Accomplished. The four expression plasmids (pZR2524-2527) had been successfully constructed during 0/01/2024 to 03/31/2025. Next step, we will transform these four expression plasmids into N2-fixing cyanobacterium Anabaena sp. PCC 7120 for test of producing these 2xEarP-H6. Objective 3: Production, purification, and verification of EarP; 30% Accomplished. In the summer of 2025, we will transform these four expression plasmids pZR2524-2527) into N2-fixing cyanobacterium Anabaena sp. PCC 7120 for test of producing these 2xEarP-H6. We will also carry out small-scale fermentation to overproduce one of the four 2XEarP-H6 proteins in E. coli and purify it. Objective 4: Inactivate a cyanophycin synthetase gene to shunt Anabaena's N-flux from producing cyanophycin to the production of EarP. 40% Accomplished. Continue working on objective 4: Inactivate a cyanophycin synthetase gene to shunt Anabaena's N-flux from producing cyanophycin to the production of EarP. Alternatively, PmlI-1228bp-NheI internal fragment of all3879 from pZR674-3 will be subcloned into EcoRV and SpeI digested pZR606S (SP) to produce pZR2531. The resulting cargo plasmid pZR2531 will be transformed into Anabaena by conjugation. The cargo plasmid is integrated into Anabaena chromosome by single crossover homologous recombination to generate 3′- and 5′-deleted copies of all3879. Thus, all3879 is split into two truncated copies and thereby be inactivated (designated as SR3879). Then, the EarP plasmid with the highest EarP production/secretion will be transformed into SR3879. Finally, the EarP yield can be quantified using the methods described in Objective 3.

Impacts
What was accomplished under these goals? Objective 1: Design and synthesize the EarP gene. 100% Accomplished The endogenous protein Asr1915 identified in Anabaena annotated proteome has 60 amino acids in length containing 47 EAA (78.3%). The mini-EarP were optimized with the recommend mole ratio and the popular di-peptide bond formation using Asr1915 as a starting point. The resulting sequences designated EarP that contains 100% EAA. Two identical copies of EarP (2xEarP) were used to generate their codon optimized, different nucleotide sequence based on Anabaena's codon usage table. The 2xEarP's nucleotide sequences are designed and synthesized at IDT. Objective 2: Construct expression plasmids for EarP over-production and secretion. 65% Accomplished To overexpress and purify the EarP protein, the synthetic different versions of EarP gene, the 2x earP codon optimized gene with a six-histidine tag (H6) at C-terminal, designed 2x EarP-H6, then different signal peptide sequences (SPS) were fused to the N-terminal of 2x EarP-H6. The four signal peptide sequences are Gene3SPS, All4499SPS, Alr4550SPS and Alr0608-1SPS. These peptide sequences are fused to the proper positions of 2xEarP and the 2XearP genes are synthesized at IDT. The synthesized genes are subcloned into NdeI-SalI digested pET28a to produce pZR2511, 2512,2513,2514. These four synthesized genes were successfully expressed in E. coli BL21DE3. All four SPS-2xEarP-H6 protein expressions were confirmed by Western blot using the penta-his tag antibody (Qiagen). These were exciting results; all the designed proteins (not present in nature) are successfully synthesized in E. coli. During this project period 04/01/2024 to 03/31/2025, we were focusing on subcloning these genes from E. coli's expression vector pET28a into Anabaena expression vector pZR1188 to produce pZR2524, 2525, 2526, 2527 for overproducing these 2xEarP-H6 in N2-fixing cyanobacterium Anabaena sp. PCC 7120. The four expression plasmids (pZR2524-2527) have been successfully constructed. After verification by PCR and/or DNA sequencing, next step, we will transform these four expression plasmids into N2-fixing cyanobacterium Anabaena sp. PCC 7120 for test of producing these 2xEarP-H6. Objective 3: Production, purification, and verification of EarP. 30% Accomplished In the summer of 2025, we will transform these four expression plasmids pZR2524-2527) into N2-fixing cyanobacterium Anabaena sp. PCC 7120 for test of producing these 2xEarP-H6. We will also carry out small-scale fermentation to overproduce one of the four 2XEarP-H6 proteins in E. coil and purify it. Objective 4: Inactivate a cyanophycin synthetase gene to shunt Anabaena's N-flux from producing cyanophycin to the production of EarP. 40% Accomplished Briefly, PCR amplified 2706bp of all3879 coding region by primers ZR27, 28 was cloned intonpTOPO2.1 vector to produce pZR674-3, then AflII to delete 831bp (bp1319 to bp 2150), then recirculated to produce pZR675-8 (AmpKm), The pZR675-8 is ready for conjugatively transforming into Anabaena sp. PCC7120 to inactivate all3879.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Nawaz T, S Fahad, L Gu, L Xu, R Zhou (2025) Harnessing Nitrogen-Fixing Cyanobacteria for Sustainable Agriculture: Opportunities, Challenges, and Implications for Food Security. Nitrogen 2025, 6(1), 16; https://doi.org/10.3390/nitrogen6010016
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Nawaz T, L Gu, J. Gibbons, Z Hu, R Zhou (2024) Bridging Nature and Engineering: Protein-Derived Materials for Bio-Inspired Applications. Biomimetics (IF3.8); 9(6), 373; https://doi.org/10.3390/biomimetics9060373
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Nawaz T, S Fahad, S Saud, R Zhou et al (2024) Sustainable nitrogen solutions: Cyanobacteria-powered plant biotechnology for conservation and metabolite production. Current Plant Biology (IF5.6); 40 (2024) 100399; https://doi.org/10.1016/j.cpb.2024.100399.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Nawaz T, N Joshi, D Nelson, S Saud, Zhou R et al (2024) Harnessing the Potential of Nitrogen-Fixing Cyanobacteria: A Rich Bio-Resource for Sustainable Soil Fertility and Enhanced Crop Productivity. Environmental Technology & Innovation (IF6.7), Nov. 2024 DOI: 10.1016/j.eti.2024.103886
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Nawaz T, Gu L, Hu Z, S Fahad, S Saud, Zhou R (2024) Advancements in Synthetic Biology for Enhancing Cyanobacterial Capabilities in Sustainable Plastic Production: A Green Horizon Perspective. Fuels (IF2.7), 5, 394438. https://doi.org/10.3390/fuels5030023
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Nawaz T, Liping Gu L, S Fahad, S Saud, Bleakley B, Zhou R (2024) Exploring Sustainable Agriculture with Nitrogen-Fixing Cyanobacteria and Nanotechnology. Molecules (IF4.6), May 28;29(11):2534 doi: 10.3390/molecules29112534.

Progress 04/01/23 to 03/31/24

Outputs
Target Audience:Companies involved in Biosynthesis and Bioproduction such as Houdek, South Dakota Innovation Partners (SDIP), CyanoSun Energy and animal feeds companies. These companies form the base of our private sector partnerships and will provide the most direct route to commercialization. Genetic engineering of N2-fixing cyanobacteria has been incorporated into two existing high-level courses (MICR 450/550-Biotechnology; MICR 438L-Molecular Biology Lab) which the PI has been teaching. The target audiences include undergraduate students, graduate students, postdocs/visiting scientists, and higher schoolteachers/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 two existing courses that PI/Co-PI teaches: Micro 450/550, Applied Microbiology and Biotechnology; and MICR 438L-Molecular Biology Lab. There were 15 students enrolled in MICR450/550; and 9 students in MICR438L during the report period. During 2023-2024, a total of 4 personnel received hands-on research training in molecular biology and biotechnology from this grant. These included two PhD students: Taufiq Nawaz and Dillon Nelson, one visiting scientist Dr. Shah Fahad, and one high school student Kyle Tan. 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. One review paper titled "Sustainable protein production through genetic engineering of cyanobacteria and use of atmospheric N2 gas" has been published in Food and Energy Security. What do you plan to do during the next reporting period to accomplish the goals?1) We will wrap up objective One: Design and synthesize the EarP gene. Specifically, we will synthesize Asr1915 gene in IDT. Asr1915 coding for an endogenous putative protein that has 60 amino acids in length containing 47 EAA (78.3% EAA) will be overexpressed in E. coli and Anabaena. 2) Continue working on Objective 2: Construct expression plasmids for EarP over-production and secretion. Next step, we will subclone these genes into Anabaena expression vector pZR1188 to overproduce these 2xEarP-H6 in N2-fixing cyanobacterium Anabaena sp. PCC 7120. 3) Continue working on objective 3: Production, purification, and verification of EarP. We will subclone these genes into Anabaena expression vector pZR1188 to overproduce these 2xEarP-H6 in N2-fixing cyanobacterium Anabaena sp. PCC 7120 and purify them using cobalt-based resin to purify these proteins. 4) Start working on objective 4: Inactivate a cyanophycin synthetase gene to shunt Anabaena's N-flux from producing cyanophycin to the production of EarP. Briefly, PCR amplified all3879 internal fragment will be cloned into the integrative vector pZR606 The resulting cargo plasmid will be transformed into Anabaena by conjugation. The cargo plasmid is integrated into Anabaena chromosome by single crossover homologous recombination to generate 3′- and 5′-deleted copies of all3879. Thus, all3879 is split into two truncated copies and thereby be inactivated (designated as SR3879). Then, the EarP plasmid with the highest EarP production/secretion will be transformed into SR3879. Finally, the EarP yield can be quantified using the methods described in Objective 3.

Impacts
What was accomplished under these goals? Goal One: Design and synthesize the EarP gene. 90% Accomplished The endogenous protein Asr1915 identified in Anabaena annotated proteome has 60 amino acids in length containing 47 EAA (78.3%). The mini-EarP were optimized with the recommend mole ratio and the popular di-peptide bond formation using Asr1915 as a starting point. The resulting sequences designated EarP that contains 100% EAA. Two identical copies of EarP (2xEarP) were used to generate their codon optimized, different nucleotide sequence based on Anabaena's codon usage table. The 2xEarP's nucleotide sequences are designed and are synthesized at IDT. Objective 2: Construct expression plasmids for EarP over-production and secretion. 50% Accomplished To overexpress and purify the EarP protein, the synthetic different versions of EarP gene, the 2x earP codon optimized gene with a six-histidine tag (H6) at C-terminal, designed 2x EarP-H6, then different signal peptide sequences (SPS) were fused to the N-terminal of 2x EarP-H6. The four signal peptide sequences are Gene3SPS, All4499SPS, Alr4550SPS and Alr0608-1SPS. These peptide sequences are fused to the proper positions of 2xearP and are synthesized at IDT. The synthesized genes are subcloned into NdeI-SalI digested pET28a to produce pZR2511, 2512,2513,2514. These four synthesized genes were successfully expressed in E. coli BL21DE3. All four SPS-2xEarP-H6 protein expressions were confirmed by Western blot using the penta-his tag antibody (Qiagen). These are exciting results; all the designed proteins (not present in nature) are successfully synthesized in E. coli. Next step, we will subclone these genes into Anabaena expression vector pZR1188 to overproduce these 2xEarP-H6 in N2-fixing cyanobacterium Anabaena sp. PCC 7120. Objective 3: Production, purification, and verification of EarP. 10% Accomplished In summer of 2024, we will subclone these genes into Anabaena expression vector pZR1188 to overproduce these 2xEarP-H6 in N2-fixing cyanobacterium Anabaena sp. PCC 7120. Objective 4: Inactivate a cyanophycin synthetase gene to shunt Anabaena's N-flux from producing cyanophycin to the production of EarP. 0% Accomplished We will start working on objective 4 in the fall of 2024.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Nawaz T, Gu L, Fahad S, Saud S, Harrison MT, Zhou R (2024) Sustainable protein production through genetic engineering of cyanobacteria and use of atmospheric N2 gas. Food and Energy Security, https://doi.org/10.1002/fes3.536