Progress 05/15/16 to 05/14/17

Outputs
Target Audience:Companies such as South Dakota Innovation Partners(SDIP),Poet and Dakota Ethanol. 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 (MICR-438-Molecular Biology Lab; Biotechnology 450/550; ABS705/Molecular Cloning Section) which the PI has been teaching, the target audiences are extended to undergraduate students andgraduate students. The target audience also includes researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has served as an excellent example of integrating research and education. The project improved state-of-the-art in synthetic biology, photobioreactor process control, and product recovery via low cost resin-based separation. The knowledge and infrastructure supporting this platform project has been used in an existing course (Micr 450/550, Applied Microbiology & Biotechnology; Micr 438L-Molecular Biology Lab) that PI has been teaching for years. The PI Dr. Zhou also taught several lectures in a lab-based graduate course ABS 705 using the project generated knowledge. From 05/15/2016 to 05/14/2017, a total of 3 undergrad students (Matthew McKillop, Trevor VanDenTop, and Tanner Wetzel), four graduate students (Jaimie Gibbons-Ph.D., Yeyan Qiu-Ph.D., Chuck Halfmann-Ph.D. and Nathanael Braselton-M.S.), one visiting scientist (Dr. Shengni Tian) and one technician (Huilan Zhu) received training from this project. How have the results been disseminated to communities of interest?Poster Presentation: N2-Fixing Cyanobacteria Harnessed for Biosolar Production of Nitrofertilizer; FY2016 AFRI and NIWQP PD meeting at Washington Marriott at Metro Center, Washington, D.C. What do you plan to do during the next reporting period to accomplish the goals? Obj 1. Engineering Anabaena to produce and secrete ammonia The remaining activities in this objective will be integrated into Obj. 2 Obj 2. Creating a desired mutant with higher frequency of functional heterocysts We wil characterize the phenotype and N2-fixing /ammonia secretion capacity for the patA++patN-strains we created during 2016. If the patA++patN-strains do have more closely spaced heterocysts and fix more N2, they will serve as a model strain for further genetic modification to produce more ammonia. We will create an Anabaena patN-patB++mutant.Inactivation of patN gene in Nostoc punctiforme produced a mutant with closer spaced singular heterocysts, with an average vegetative cell interval of 3-4 cells compared to 12-13 for the wild type. However, the patN mutant has lower nitrogen fixing capability. Our hypothesis is that overexpression of PatB, the specific transcription factor required for nif genes expression in heterocysts, may increase the capability of N2-fixing in patN-patB++ mutant. We will create a mutant with simultaneous inactivation of patN (alr4812) and ectopic expression of PglnA-PpatB-patB in Anabaena patN. Obj 3. Genetic shunting Anabaena's nitrogen flow from producing nitrogen-enriched storage polymers to production of excreted ammonia We will shut down cyanophycin synthesis in the best ammonia-secreting strain M2803 strain. Many cyanobacteria produce N-enriched cyanophycins, which are co-polymers of aspartate and arginine produced through non-ribosomal pathways. By circumventing these storage granules, the N2-derived ammonia flux can be redirected for ammonia excretion. The synthesis of cyanophycin is through cyanophycin synthetase encoded by all3879. The gene all3879 will be knocked out through a single/double-crossover approach, which has been a routine approach in my laboratory.

Impacts
What was accomplished under these goals? Obj 1. Engineering Anabaena to produce and secrete ammonia (90% accomplished) 1) Three independent Anabaenamutants called M2083-1, M2083-2 and M2083-3have been successfully created through inactivating the glutamine synthase gene (alr2328) in Anabaena sp. PCC 7120. These three mutants are capable of growing nearly as well as the wild-type using atmospheric N2 gas as the sole nitrogen source. The mutants on average secreted 5, 3 and 20-fold more ammonia than the wild-type strain at 26, 48 and 72h, respectively. The average secretion rates of ammonia during 26h, 48h and 72h were 6.6, 44.2 and 254 µg/ml/OD700/h, respectively, measured by the LIGS-136NHM-XS Micro-Ammonia Ion Electrode System (Lazar Research Labortories Inc. Los Angeles, CA). 2) Three independent Anabaenamutants M2084-3, M2084-7, and M2084-9 have been successfully created throughoverexpressing an anti-sense internal fragment from alr2328 toknock down the glutamine synthase activity, so that M2084 will produce and secrete ammonia as well.The M2084 strains secreted 108, 16, 13-fold more ammonia than the wild-type strain at 26, 48 and 72 hours, respectively. The average secretion rates of ammonia during 26h, 48h and 72h were 145, 220.1 and 154 µg/ml/OD700/h, respectively. Obj 2. Creating a desired mutant with higher frequency of functional heterocysts We combined inactivating patN and overexpressing patA in the wild type to create a desired mutant with a closer spaced singular heterocysts. Overexpression of patA gene in Anabaena led to 2-fold increase of multiple singular heterocysts. Also, inactivation of patN gene in Nostoc punctiforme produced a mutant with closer spaced singular heterocysts, with an average vegetative cell interval of 3-4 cells compared to 12-13 for the wild type. We created such an Anabaena mutant by over-expression of patA in Anabaena-patN mutant strains patN11242, patN12156 (Dr. Wolk 's gift). These six patA++patN-strains were verified by colony PCR. They were named patN11242(pZR2177)-1,-2,-3; patN12154(pZR2177)-1, -2, -4. They are predicted to have more closely spaced heterocysts and might fix more N2. The phenotype for patA++patN-strain and its N2-fixing capacity are being characterized. If successful, the patA++patN-strain will serve as a model strain for further genetic modification to produce more ammonia. Obj 3. Genetic shunting Anabaena's nitrogen flow from producing nitrogen-enriched storage polymers to production of excreted ammonia Many cyanobacteria produce N-enriched cyanophycins, which are co-polymers of aspartate and arginine produced through non-ribosomal pathways. By circumventing these storage granules, the N2-derived ammonia flux can be redirected for ammonia excretion. The synthesis of cyanophycin is through cyanophycin synthetase encoded by all3879. Inactivating the gene all3879 in Anabaena is in progress.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: 1) Poster Presentation: N2-Fixing Cyanobacteria Harnessed for Biosolar Production of Nitrofertilizer; FY2016 AFRI and NIWQP PD meeting at Washington Marriott at Metro Center, Washington, D.C. 2) Invited talk titled Cyanobacteria: The Best Organisms Under the Sun at Gamma Sigma Delta Annual Awardee Seminar of South Dakota State University, 04/03/2017.

Progress 05/15/15 to 05/14/16

Outputs
Target Audience:Companies such as South Dakota Innovation partnerships (SDIP),VeraSun Energy. 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 (MICR438-Molecular Biology Lab; MICR450/550-Biotechnology; ABS705/Molecular Cloning) which the PI has been teaching, the target audiences are extended to undergraduate students and graduate students. Changes/Problems:We modified the proposed Task 1 (co-express Anabaena's antisense glnA and an E. coli ammonia transporter gene amtB in Anabaena) to current Task 1: Over-express Anabaena's antisense glnA in Anabaena sp. PCC7120 or related mutants which we created in this project. We will not over-express an E. coli ammonia transporter gene amtB in Anabaena becausethatat least four presumative homologues genes coding forammonia transporter (AmtB) are identifiedin the genome of Anabaena sp. PCC7120. They are alr0990,alr0991, alr0992 and alr4682. Instead of overexpressing an E. coli ammonia transporter gene amtB in Anabaena,we are inactivating these four ammonia transporter genes to determine their roles in this N2-fixing Anabaena strainsp. PCC7120. What opportunities for training and professional development has the project provided?This project has served as an excellent example of integrating research and education. The project improved state-of-the-art in synthetic biology, photobioreactor process control, and product recovery via low cost resin-based separation. The knowledge and infrastructure supporting this platform project has been used in an existing course (Micr 450/550, Applied Microbiology & Biotechnology; Micro438L-Molecular Biology Lab) thatPI has been teaching for years.The PI Dr. Zhou also taught several lectures in a lab-based graduate course ABS 705 using the project generated knowledge. From 05/15/2015 to 05/14/2016, a total of2 undergrad students, five graduate students, two visiting scientists and one technician received training from this project. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?In next reporting period, 1) we will finish measurring the ecretion of ammoniafrom SR2083 and A7120(pZR2084) and characterizing the phenotype for the patA++patN-strain we created during year 2015; 2) We will focus on below Task3-5. Task 3: Combined inactivating patN and overexpressing hetR in the constructed ammonia-secreting strain HetR is the master regulator of heterocyst differentiation in Anabaena and is absolutely required for positive regulation of heterocyst formation. Ectopic expression of hetR in Anabaena increases heterocyst frequency by about 2-fold, and the nitrogenase activity was also 1.5-2-fold higher than that of wild type during a period of 5 days .For task 3, we will create a mutant with simultaneous deletion of patN (alr4812) and ectopic expression of hetR (alr2339) in Anabaena. The experimental approach is similar to the construction of pZR1988 described in details in Task 2, except for replacing patA with hetR accordingly. Task 4: Shut down the cyanophycin synthesis in the best ammonia-secreting strain Task 5: Assess and recover the continuous ammonia production in flask culture

Impacts
What was accomplished under these goals? 1) The glutamine synthase gene (alr2328)has beeninactivated through a single cross-over recombination (SR). This involves ligating the internal alr2328 fragment (910bp) into the pZR606 integrationvector to produce pZR2083, then introducingpZR2083 into Anabaena sp. PCC7120. This is designed to knock out alr2328 in Anabaena sp. PCC7120 to create an Anabaenamutant named SR2083to produce and secrete ammonia.Measuring the secretion of ammoniafrom SR2083 strain is underway. 2) An anti-sense internal fragment from alr2328 was fused to the glnA (alr2328) promoter that was previously constructed in a replicating vector pZR670 for Anabaena sp. PCC7120to produce pZR2084. pZR2084 was successfully transformed into Anabaena sp. PCC7120 to create an Anabaenamutant named SR2084toknock down the glutamine synthase activity, so that we expect that A7120(pZR2084) strain will produce and secrete ammonia as well.Measuring the secretion of ammoniafrom A7120(pZR2084) strain is underway. 3) Combined inactivating patN and overexpressing patA in Anabaena sp. PCC7120to create a desired mutant with a closer spaced singular heterocysts. Overexpression of patA gene in Anabaena led to 2-fold increase of multiple singular heterocysts. Also, inactivation of patN gene in Nostoc punctiforme produced a mutant with closer spaced singular heterocysts, with an average vegetative cell interval of 3-4 cells compared to 12-13 for the wild type. We have created an Anabaena mutant by over-expression of patA in Anabaena-patN mutant strain (Dr. Wolk 's gift) . This patA++patN-strain, predicted to have more closely spaced heterocysts and might fix more N2. The phenotype for patA++patN-strain and its N2-fixaing capacity are being characterized. If successful, the patA++patN-strain will serve as a model strain for further genetic modification to produce ammonia.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Kangming Chen, Huilan Zhu, Liping Gu, Shengni Tian, and Ruanbao Zhou, 2016. Target Gene Inactivation in Cyanobacterium Anabaena sp. PCC 7120, Bio-Protocol