Progress 11/01/09 to 09/30/14
Outputs
Target Audience:Companies such as ICM, Inc, South Dakota Innovation partnerships (SDIP),VeraSun Energy, Separation Kinetics, 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 two existing high level courses (Biotechnology 450/550; ABS705/Molecular Cloning Section) which the PI has been teaching, the target audiences are extended to undergraduate students,graduate students, posdocs and visiting scientists. Changes/Problems:Because we did not obtain the MBO synthesis gene from our collaborators in the initiation stage of the project, in the first annual report-2010 annual report, we had to modify our project by replacing our model product methylbutenol (C5) with long- chain hydrocarbons such as C10 and C15 alkenes. We were also allowed to change the title of our project to be ENGINEERING CYANOBACTERIA AS A FACTORY TO PRODUCE BIOFUELS AND HIGH VALUE CHEMICALS. 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, photobioreactor process control, and product recovery via low cost phase separation. The knowledge and infrastructure supporting this platform project has been used in an existing course (Micr 450/550, Applied Microbiology & Biotechnology) that Drs. Zhou, Gibbons have been teaching, and developing a new graduate course (Molecular Metabolic Engineering or Synthetic Biology) that Dr. Zhou has already taught several lectures in a lab-based graduate course ABS 705. Dr. Zhou also developed a new lab-based course MICR438L-Molecular Biology Lab in fall semester of 2014. Just in 2014, total 21 personnel (two faculty members, two Ph.D. students, three MS graduates, five undergrads, six high school teachers, three postdocs) received education training or professional development from this project. How have the results been disseminated to communities of interest?(1) The PI had presented the research findings at the NC-Sun Grant Annual Meetings (2010-2014), and the PI had been invited to give a talk on BIT's 3rd Annual World Congress of Bioenergy (April25-27, 2013, Nanjing, China). (2) More than 20 posters/oral presentations from my students were presented in either International Workshop on Cyanobacteria (Washington University in St. Louis, August 7-11, 2013), or in several National/Regional Scientific Meetings. (3) Additionally, Metabolic Engineering Cyanobacteria has been incorporated to three existing high level courses (Biotechnology 450/550; ABS705/Genetic Engineering; MICR438L-Molecular Biology Lab) which the PI has been teaching, the target audiences are extended to undergraduate students, graduate students and higher school students &teachers. (4) In 2012, 2014, the PI co-hosted (with Dr. Xingyou Gu) the One Week-Plant Molecular Biology workshop for the higher school students &teachers from State of South Dakota. Starting 2013, every year, the PI has been invited to give a guest lecture "Synthetic Biology of Cyanobacteria for Biofuels" to about100 undergrad students in General Botany Bot201 class. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
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 (data shown in the report of June, 2013).The first generation of transgenic cyanobacteria has been confirmed to produce and secret a significant amount of myrcene. Then, we constructed plasmid pZR1463 that contains DXP operon. This synthetic operon is designed to pull more carbon flux to produce GPP, the precursor for myrcene. The second generation strain bearing pZR1463 produces only slightly more myrcene than the first generation strain bearing pZR966. Since our previous work on limonene production, we observed a 6.8-fold increase in limonene yield when expressing the DXP operon in conjunction with limonene synthase, compared to limonene synthase alone. We concluded that the myrcene synthase is limiting factor. To further increase the myrcene productivity, we started to look for a presumptive better myrcene synthase gene from the myrcene-producing plant thyme, in which the leaves of thyme was reported to contain up to 40% by weight of myrcene (Behr & Johnen,2009 ChemSusChem). Cloning myrcene synthase gene from thyme is underway. (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 also succeeded in engineering Anabaena sp. PCC7120 to produce bisabolene (C15H24) using air, mineralized water and sunlight (manuscript in preparation). (5)We also analyzed linalool accumulation in Anabaena harboring pLinS (LinS Anabaena) and pLinS-DXP (LinS-DXP Anabaena) over a 14-day growth period in four growth conditions: in BG11 media supplemented with combined nitrogen (+N) and without (-N), in combination with 50 µE·m-2·s-1 light (low light) and 150 µE·m-2·s-1 (high light). We found that LinS-DXP Anabaena produced the most linalool (353.5 ± 38.6 µg·L-1) during the growth trial when supplemented with combined nitrogen (+N) in low light conditions. This total production dropped nearly 40% when grown in -N conditions. We attribute this lower production to a slower growth rate due to a lack of combined nitrogen in the growth media. This would increase the time of lag phase, due to the cells investing energy and resources into nitrogen fixation. Although linalool production from LinS-DXP Anabaena was lower in -N compared to +N conditions during the first four days of the growth trial, we observed production increase every two days, until the production rate between +N and -N conditions was similar during the last two days of the growth period. We hypothesize that the culture initially has lower production due to a longer lag phase, but after nitrogen fixation is established, the cells go into exponential growth and "catch up" to the culture grown in +N. In summary, the major goal of this project is to engineering cyanobacteria as a factory to produce biofuels and high value chemicals using solar energy. We very well accomplished the major goal of this project. That is, we successfully engineered N2-fixing cyanobacterium Anabaena sp. PCC7120 to produce linalool (C10H18O), myrcene (C10H16), Limonene (C10H16), farnesene (C15H24), and bisabolene (C15H24) using only air (N2 gas &CO2), mineralized water and sunlight.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Halfmann C, L Gu, W Gibbons, R Zhou* (2014) Metabolic engineering of a cyanobacterium to convert CO2, water, and light into a long-chained alkene. Paper number SD14-050 (doi: 10.13031/sd14050), Published by the American Society of Agricultural and Biological Engineers
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Johnson TJ, M Hildreth, L Gu, R Zhou, and W Gibbons (2014) Utilizing a Dual-Fluorescence Assay to Quantify Viability of Filamentous Cyanobacteria. Paper Number: SD14-048 (http://elibrary.asabe.org/azdez.asp?JID=8&AID=44318&CID= smpnc&T=2). Published by the American Society of Agricultural and Biological Engineers
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Zhou R*, OA Koksharova (2014) HepK, a protein-histidine kinase from the cyanobacterium Anabaena sp. strain PCC 7120, binds sequence-specifically to DNA. Trends in Bacteriology. 2014
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Halfmann C, L Gu, W Gibbons, and R. Zhou* (2014) Genetic Engineering Cyanobacteria to Convert CO2, Water and Light into the Long-Chain Hydrocarbon Farnesene. Appl Microbiol Biotechnol. 98:98699877
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Halfmann C, L Gu, and R. Zhou* (2014) Engineering Cyanobacteria for Production of a Cyclic Hydrocarbon from CO2 and H2O. Green Chem. 16 (6), 3175 3185.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Chen K, X Xu, L Gu, and R Zhou* (2014). Simultaneous Gene Inactivation and Promoter Reporting in Cyanobacteria. (Accepted in Applied Microbiology and Biotechnology
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2014
Citation:
Kangming Chen, 2014. Ph.D. Dissertation title: REGULATED INTRAMEMBRANE PROTEOLYSIS IN ANABAENA VARIABILIS
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2014
Citation:
Charles T. Halfmann, 2014. MS Thesis title: Engineering Cyanobacteria for Production of a Cyclic Hydrocarbon from CO2 and H2O.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Halfmann C, L Gu, and R Zhou (2014) Engineering N2-fixing cyanobacteria for photosynthetic production of limonene. p14, in the 1st International Plant Synthetic Biology workshop, May 17-18th, 2014 at MIT, Boston.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Halfmann C, L Gu, and R Zhou (2014) Photosynthetic production of a cyclic hydrocarbon by an engineered cyanobacterium. B35, in Abstracts of Ninth Annual DOE Joint Genome Institute User Meeting. Mar 18-20th, 2014 Walnut Creek, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Chen K, R Zhou (2014) Ava_4785, a site-2 intramembrane metalloprotease, is required for cold acclimation in Anabaena variabilis. P175, in the abstracts of 2014 Molecular Genetics of Bacteria and Phages Meeting. University of Wisconsin-Madison, Aug 5-9th, 2014.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Xinyi Xu, Liping Gu, Ruanbao Zhou (2014) Characterization of Five Putative Aspartate Aminotransferase Genes in Anabaena sp. PCC 7120. Departmental Research & Scholarship Day, April, 2014.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Four Undergrad Research Poster Presentations (Jordan Neises, Kelly Sutko, Kristen Kludt, Bryan Mejia-Sosa) at Departmental Research & Scholarship Day, April/Nov., 2014.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Engineering N2-fixing cyanobacteria to photosynthetically produce a long-chain hydrocarbon-myrcene. Talk at Annual NC-Sun Grant Meeting , Mar 15-17,2014;
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
Zhou, R. (2014) Synthetic Biology of Cyanobacteria for Biofuel Production to Bot 201-General Botany. Feb, 16, 2014
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Companies such as ICM, Inc, South Dakota Innovation partnerships (SDIP), Cyanosun Energy, VeraSun Energy, Separation Kinetics are developing biomass to ethanol processes. These companies form the base of our private sector partnerships and might provide the most direct route to commercialization. Metabolic engineering cyanobacteria has been incorporated to two existing high level courses (Biotechnology 450/550; ABS705-Metabolic Engineering Section) which the PI has been teaching, the target audiences are extended to undergraduate students and graduate students. Changes/Problems: No significant project modification in 2013 although we started working on engineering of cyanobacteria to produce other hydrocarbons such as myrcene, limonene, farnesence, methylbutenol, ethylene etc. What opportunities for training and professional development has the project provided? In the 2013, there were 14 people involved in this project. Two faculty Dr. Ruanbao Zhou and Dr.Liping Gu from Department of Bio-Microbiology have been working on engineering cyanobacteria to produce drop-in fuels or chemicals. The two postdocs, Drs. Xinyi Xu (08/2012-present), Huilan Zhu(07/2012-present) and two graduate students (Chuck Halfmann, Nate Braselton) were also partially working on this project. Eight undergraduate students (Jaimie Gibbons, Ashley LaCayo, Melissa Livermont, William Uhlemann, Megan Quast, Aldon Myrlie, Jacob Sobraske, Ashley Harris) participated in this research and received hands-on experience on metabolic engineering of cyanobacteria. How have the results been disseminated to communities of interest? The PI had presented the research findings at the NC-Sun Grant Annual Meetings (March 26-27, 2013, Chicago, IL), and the PI had been invited to give a talk on BIT's 3rd Annual World Congress of Bioenergy (April25-27, 2013, Nanjing, China). Six posters from my students were presented in 11th International Workshop on Cyanobacteria (Washington University in St. Louis, August 7–11, 2013); Four oral presentations and two poster presentations at the 73rd Annual Meeting North Central Branch of the ASM, October 11 and 12, 2013, South Dakota State University, Brookings. Additionally, Metabolic Engineering Cyanobacteria has been incorporated to two existing high level courses (Biotechnology 450/550; ABS705/Metabolic Engineering) which the PI has been teaching, the target audiences are extended to undergraduate students and graduate students. What do you plan to do during the next reporting period to accomplish the goals? The major goal for next reporting period is focused on Metabolic Flux Modifications to significantly increase the productivity for our target fuel molecules such as linalool, limonene, myrcene, methylbutenol/isoprene etc; To do so, 1) We will overexpress a rate-limiting bi-functional enzyme (FBP/SBPase) for regeneration of RuBP (acceptor for CO2) in our linalool-producing Anabaena strain to boost the RuBP level and thus subsequently increase CO2 fixation. 2) Increase the fixed carbon flow to synthesis of GPP (the direct precursor for linalool, limonene and myrcene) by overexpressing a synthetic operon coding for the three limiting enzymes (DXS-IDI-GppS) required for synthesis of GPP. 3) Blocking glycogen synthesis to redirect the newly fixed carbon flux to synthesis of our target fuel molecules, instead of storage glycogen or biomass. In addition to conducting this biofuel related research, I will also initiate two new projects: 1) Genome-wide study of regulated intramembrane proteolysis in Anabaena; 2) Genetic study of akinete/heterocyst differentiation in Anabaena cylindrica.
Impacts
What was accomplished under these goals?
In the past year, we made substantial progress on this project. 1) We developed a resin-based recovery method for the volatile linalool produced by engineered cyanobacteria. A small glass column filled with 100 mg of Supelpak 2SV resin (Supelco) was attached to the exhaust port of each flask to capture the volatile linalool and other volatile metabolites from the culture headspace. Resin samples from each flask were washed twice with 2.5 ml pentane containing 1 µg mL-1 tetracosane as an internal standard (IS), pooled in 5 mL total volumes, evaporated to 1 mL using gentle stream of N2 gas, and subjected to gas chromatography-mass spectrometry (GC-MS) analysis of linalool productivity. 2) We have successfully transferred a plant myrcene synthase (MyrS) gene into cyanobacterium Anabaena sp. PCC7120. The resulting transgenic cyanobacterium was confirmed to produce myrcene (C10H16) by GC-MS. 3) Since the yield of linalool from the 1st generation of genetically engineered strain is quite low, so we have been focusing on improving the linalool productivity. Competition among different pathways for the newly-fixed carbon flux, including storage carbohydrate (mainly glycogen) biosynthesis, may limit carbon flux to linalool production. Therefore, reducing carbon flux to the competitive glycogen pathway is necessary for boosting linalool production. To this end, we had knocked out the gene all4645, the only one gene encoding ADP-glucose pyrophosphorylase in Anabaena genome, which controls the first step reaction of glycogen synthesis. Thus, more carbon flow in all4645 knockout (KO) strain will be redirected to production of our target products. After we obtain a complete segregated strain, we will transform this strain with linalool synthase construct pLinS for linalool production. We will assess if this knockout strain significantly improves the production of linalool. 4) We had constructed an expression plasmid pZR1464 for overexpressing a synthetic operon to boost the endogenous GPP level. This operon encodes for three limiting enzymes required for synthesis of GPP in MEP pathway. pZR1464 has been transferred into a linalool-producing cyanobacterial strain for assessing the linalool production. 5) We also have started working on engineering cyanobacteria to produce other hydrocarbons such as isoprene, methylbutenol, ethylene, limonene.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
1. Zhang Y., P. Luethy, R. Zhou, and Lee Kroos. 2013. Residues in Conserved Loops of Intramembrane Metalloprotease SpoIVFB Interact with Residues near the Cleavage Site in Pro-?K. J Bacteriol. 195(21):4936-4946.
2. Halfmanna C., L. Gu, and R. Zhou 2013. Engineering Cyanobacteria for Production of a Cyclic Hydrocarbon from CO2 and H2O. (submitted to Green Chemistry on 12/19/2013).
3. Zhou R., K. Chen, X. Xiang, L. Gu and L. Kroos. 2013. Features of Pro-?K important for cleavage by SpoIVFB, an intramembrane metalloprotease. J. Bacteriol. 195(12):2793-806).
4. Gu L., X. Xiang, D. Raynie, W. Gibbons, R. Zhou. 2013. Biosolar conversion of H2O and CO2 into linalool by engineered cyanobacteria. The Proceedings of 2012 Sun Grant National Conference (Published online http://sungrant.tennessee.edu/NR/rdonlyres/DDF120E1-C312-4065-B095-6EC87BD11DA8/3651/318Zhou_Ruanbao.pdf).
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: OUTPUTS: Our research goal is to develop a suite of engineered cyanobacterial strains that are separately capable of directly converting CO2 and H2O into biofuels and commodity chemicals using free solar energy. This will improve resource-use efficiency of biorefineries by recovering CO2 lost from fermentation and improve economic vitality by converting this unused CO2 back to a drop-in fuel or high value chemicals. This proposal is to take advantage of using the highest photosynthetic efficiency of cyanobacteria, which provides a greater MEP pathway flux to make the precursor for production of both long-chain hydrocarbons and long-chain alcohols. Four objectives are 1) genetically shunt the native isoprenoid biosynthesis pathway (MEP pathway) of cyanobacteria to produce long-chain alcohols and hydrocarbons by transferring-in the corresponding genes; 2) maximize production and excretion of the target products by blocking or reducing the carbon flow to competing pathways; 3) Identify long-chain hydrocarbons (alkenes/alkanes) innately produced by nitrogen-fixing cyanobacteria; 4) Identify the genes required for biosynthesis of such long-chain alkens/alkanes. Dissemination: The PI had presented the research findings at the NC-Sun Grant annual Meetings (01/2012), and at 2012 Sun Grant National Conference in New Orleans (Oct. 2-5, 2012). The PI has been invited to give a talk on BIT's 3rd Annual World Congress of Bioenergy, China, 2013. Two posters from my students were presented in 2012 NC-ASM annual meetings. Metabolic engineering cyanobacteria has been incorporated to two existing high level courses (Biotechnology 450/550; ABS705/Molecular Cloning Section) which the PI has been teaching, the target audiences are extended to undergraduate students and graduate students. PARTICIPANTS: In the 2012, there were 11 people involved in this project. Dr. Ruanbao Zhou and Dr.Liping Gu from Department of Bio-Microbiology has been working on engineering cyanobacteria to produce drop-in fuels or chemicals. The postdocs Xianling Xiang (03/2011- 03//2012),Xinyi Xu (08/2012-present), Huilan Zhu(07/2012-present) were also partially working on this project. Six undergraduate students (Jaimie Gibbons, Jessikah Moutray, Ashley LaCayo, Melissa Livermont, Israel Worthington, Megan Byram) participated in this research and received hands-on experience on metabolic engineering of cyanobacteria. TARGET AUDIENCES: Companies such as ICM, Inc, South Dakota Innovation partnerships (SDIP),VeraSun Energy, Separation Kinetics, 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 two existing high level courses (Biotechnology 450/550; ABS705/Molecular Cloning Section) which the PI has been teaching, the target audiences are extended to undergraduate students and graduate students. PROJECT MODIFICATIONS: No significant project modification in 2012 although we started working on engineering of cyanobacteria to produce other hydrocarbons such as myrcene,limonene,isoprene, methylbutenol,ethylene etc.
Impacts
In the past year, we made substantial progress on this project. 1) The linalool synthase gene from a linalool-emitting plant was fused to Anabaena promoters and subcloned into a shuttle vector. This construct was transformed into Anabaena, resulting in expression of the plant linalool synthase. This transgenic Anabaena had been confirmed to produce and secrete linalool. Our engineered cyanobacteria now can convert unutilized CO2 into linalool, driven by solar energy. Altering cyanobacteria's metabolism to directly produce end product (instead of intermediates such as lipids or polysaccharides/biomass) eliminates subsequent conversion processes that are required in current biofuel production systems. 2) We have successfully transferred a plant myrcene synthase (MyrS) gene into cyanobacterium Anabaena sp. PCC7120. The plant MyrS was expressed in Anabaena PCC7120. But we have not detected any myrcene production by this transgenic Anabaena. 3) In addition to this finding, we also made some progress on objective 3. We discovered that N2-fixing Anabaena is innately capable of producing and secreting long-chain alkanes/alkenes (C9-C18). This new finding will serve as a future research thrust to identify the alkane synthetic pathway, and then manipulate it for alkane production. 4) since the yield of linalool from the 1st generation of genetically engineered strain is quite low, so we have been focusing on improving the linalool productivity. Competition among different pathways for the newly-fixed carbon flux, including storage carbohydrate (mainly glycogen) biosynthesis, may limit carbon flux to linalool production. Therefore, reducing carbon flux to the competitive glycogen pathway is necessary for boosting linalool production. To do so, we are working on knocked out the gene all4645, the only one gene encoding ADP-glucose pyrophosphorylase in Anabaena genome, which controls the first step reaction of glycogen synthesis. Thus, more carbon flow in all4645 knockout (KO) strain may be redirected to production of our target products. After we obtain a complete segregated strain, we will transform this strain with linalool synthase construct pLinS for linalool production. We will measure if this knockout strain significantly improves the production of linalool. 5) we also have started working on engineering cyanobacteria to produce other hydrocarbons such as isoprene, methylbutenol, ethylene, myrcene, limonene. Impacts: Our process will convert fermentation-derived CO2 into biofuels or high-value chemicals, increasing the profitability of biorefineries, with no additional feedstock cost. Engineering a cyanobacterium to directly convert CO2 to excreted end products, which bypasses the expensive, multiple unit operated biomass pathway currently used in cellulosic biofuels or algal oil production. This system will recycle water in the process, not use/generate hazardous/toxic substances, and reduce CO2 emissions by 2,000 tons for every million gallons of ethanol biomass ethanol produced. The low-impact environmental footprint will foster rural economic development, and provide an excellent ROI opportunity for corn and biomass-based ethanol facilities.
Publications
- 1.Gu L., X. Xiang, D. Raynie, W. Gibbons, R. Zhou. 2012. Biosolar conversion of H2O and CO2 into linalool by engineered cyanobacteria. The Proceedings of 2012 Sun Grant National Conference (accepted).
- 2.Chen K, Gu L, Xiang X, Lynch M and Zhou R. 2012. Identification and characterization of five intramembrane metalloproteases in Anabaena variabilis J. Bacteriol. 194(22):6105-15
- 3. Halfmann C., R. Zhou. 2012. Engineering cyanobacteria to synthesize limonene from CO2 and H2O. Poster presented at 72nd NCB-ASM Annual Meeting (Fargo, ND, Oct. 12-13,2012).
- 4.Gibbons,J., R. Zhou, 2012. Identification of two genes required for synthesis of long-chain hydrocarbons in Anabaena sp. PCC7120.Poster presented at 72nd NCB-ASM Annual Meeting (Fargo, ND, Oct. 12-13,2012).
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Our research goal is to develop 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. Through purposefully genetic alteration of targeted metabolic pathways, we will redirect cyanobacteria carbon flow from producing stored bioenergy precursors (i.e., lipids and polysaccharides) to direct production of excreted products. This will enable continuous product recovery from culture fluid, while maintaining a viable cellular "factory" in a recirculating photobioreactor system. This will improve resource-use efficiency of biorefineries by recovering CO2 lost from fermentation and improve economic vitality by converting this unused CO2 back to a drop-in fuel or high value chemicals. This proposal is to take advantage of using the highest photosynthetic efficiency of cyanobacteria, which provides a greater MEP pathway flux to make the precursor for production of both long-chain hydrocarbons and long-chain alcohols. Three objectives include: 1) genetically shunt the native isoprenoid biosynthesis pathway (MEP pathway) of cyanobacteria to produce linalool and other hydrocarbons by transferring-in the corresponding genes; 2) maximize production and excretion of the target products by blocking or reducing the carbon flow to competing pathways; 3) Identify long-chain hydrocarbons (alkenes/alkanes) innately produced by nitrogen-fixing cyanobacteria; 4) Identify the genes required for biosynthesis of such long-chain alkens/alkanes. In the 2011, Dr. Zhou and one postdoc have been working on this project. This project has also provided hands-on training for three undergraduate students. The PI has presented this research findings at 2011 Alltech-North American Lecture Tour (SDSU Science Day):Feeding the world; Vet792:2011 SDSU Life Science Seminar Series; and at 2011 Annual Sun Grant Meeting. PARTICIPANTS: In the 2011,there are five people involved in this project. Dr. Ruanbao Zhou from Department of Bio-Microbiology has been working on engineering cyanobacteria to produce drop-in fuels or chemicals. The postdoc Xianling Xiang (03/2011- 12/31/2011) was also partially working on this project. Three undergraduate students (Jaimie Gibbons, Chuck Halfmann, Adeola Adebiyi) participated in this research and received hands-on experience on metabolic engineering of cyanobacteria. TARGET AUDIENCES: Companies such as ICM, Inc, South Dakota Innovation partnerships,VeraSun Energy, Separation Kinetics, 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 two existing high level courses (Biotechnology 450/550; ABS705/Molecular Cloning Section) which the PI has been teaching, the target audiences are extended to undergraduate students and graduate students. PROJECT MODIFICATIONS: No significant project modification in 2011 although we restored to work on methylbutenol(MBO).
Impacts
In the past year, we made substantial progress on above objective 1 and 3. To achieve the objective 1, the linalool synthase gene (LinS) from a linalool-emitting plant Norway Spruce was fused to Anabaena Pnir promoter and subcloned into a shuttle vector pZR807 to produce pLinS. The pLinS was transformed into Anabaena, the Western blot confirming that the plant LinS gene expressed well in Anabaena, resulting in production of linalool. A peak with a retention time of 8.2 min, found in the medium of transgenic Anabaena, was confirmed to be linalool by its mass spectra. This peak was not found in the medium of wild-type Anabaena. As expected, most of linalool produced was secreted into culture fluid. In addition to this finding, we also made some progress on objective 3. We discovered that N2-fixing Anabaena is innately capable of producing and secreting long-chain alkanes/alkenes (C9-C18). This new finding will serve as a future research thrust to identify the alkane synthetic pathway, and then manipulate it for alkane production. However, the yield of linalool from the 1st generation of genetically engineered strain is quite low. Now we are focusing on improving the linalool productivity. Competition among different pathways for the newly-fixed carbon flux, including storage carbohydrate (mainly glycogen) biosynthesis, may limit carbon flux to linalool production. Therefore, reducing carbon flux to the competitive glycogen pathway is necessary for boosting linalool production. To do so, we have knocked out the gene all4645, the only one gene encoding ADP-glucose pyrophosphorylase in Anabaena genome, which controls the first step reaction of glycogen synthesis. Thus, more carbon flow in all4645 knockout (KO) strain may be redirected to linalool production. After we obtain a complete segregated strain, we will transform this strain with linalool synthase construct pLinS for linalool production. We will measure if this knockout strain significantly improves the production of linalool. For another model product isoprene, we had a bad luck. Although we successfully transferred an IspS gene (isoprene synthase gene) into Anabaena, the transgenic Anabaena produced no detectable isoprene. Therefore we decide to discontinue working on isoprene production and restore to work on methylbutenol (MBO) production by engineered cyanobacteria. Impacts: Our process will convert fermentation-derived CO2 into linalool or isoprene, increasing the profitability of biorefineries, with no additional feedstock cost. Engineering a cyanobacterium to directly convert CO2 to excreted end products, which bypasses the expensive, multiple unit operated biomass pathway currently used in cellulosic biofuels or algal oil production. This system will recycle water in the process, not use/generate hazardous/toxic substances, and reduce CO2 emissions by 2,000 tons for every million gallons of ethanol biomass ethanol produced. The low-impact environmental footprint will foster rural economic development, and provide an excellent ROI opportunity for corn and biomass-based ethanol facilities.
Publications
- No publications reported this period
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Our goal is to develop photosynthetic cyanobacteria that are capable of converting CO2 into isoprene (C5H8) as rubber building blocks and linalool (C10H18O) as drop-in biofuels. This will improve resource-use efficiency of biorefineries by recovering CO2 lost from fermentation and improve economic vitality by converting this unused CO2 back to a drop-in fuel or chemicals. This proposal is to take advantage of using the highest photosynthetic efficiency of cyanobacteria, which provides a greater MEP pathway flux to make the precursor for production of both isoprene and linalool. Three objectives include: 1) genetically shunt the native isoprenoid biosynthesis pathway (MEP pathway) of cyanobacteria to produce isoprene and linalool by transferring-in the corresponding genes; 2) maximize production and excretion of linalool by blocking or reducing the carbon flow to competing pathways; 3) Identify long-chain hydrocarbons (alkenes/alkanes) innately produced by nitrogen-fixing cyanobacteria; 4) Identify the genes required for biosynthesis of such long-chain alkens/alkanes. Dr. Zhou and one postdoc have been working on this project.This project has also provided hands-on training for four undergraduate students. PARTICIPANTS: Dr. Ruanbao Zhou from Department of Bio-Microbiology has been working on engineering cyanobacteria to produce isoprene and linalool. The postdoc Yusheng Wu (01/2010- 08/2010) was also working on this project. Four undergraduate students received hands-on exoerience on metabolic engineering of cyanobacteria. TARGET AUDIENCES: Companies such as ICM, Inc, SDIP,VeraSun Energy, Separation Kinetics, 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. PROJECT MODIFICATIONS: Because we did not obtain the MBO synthesis gene from our collaborators, we had to modify our project by replacing our model product methylbutenol (C5H10O) with linalool (C10H18O). We also added two new objectives: 3) Identify long-chain hydrocarbons (alkenes/alkanes) innately produced by nitrogen-fixing cyanobacteria; 4) Identify the genes required for biosynthesis of such long-chain alkens/alkanes. Since we made a significant project modification, we want to change the title of our project to be ENGINEERING CYANOBACTERIA AS A FACTORY TO PRODUCE BIOFUELS AND HIGH VALUE CHEMICALS.
Impacts
Outcomes: In the past year, we made substantial progress on objective 1: genetically shunt the native isoprenoid biosynthesis pathway of cyanobacteria to produce isoprene and linalool by transferring-in the corresponding genes. Because we did not obtain the MBO synthesis gene from our collaborators, we had to change our model product to be linalool (C10H18O). Luckily, we have succeeded in engineering Anabaena to produce and secrete linalool by transferring a plant linalool synthesis gene to Anabaena. The linalool production by transgenic Anabaena was confirmed by GC/MS analysis. However, as expected, the yield of linalool from the 1st generation of genetically engineered strain is quite low. Now we are focusing on improving the linalool productivity. For another model product isoprene, we had a bad luck. Although we successfully transferred an IspS gene (isoprene synthase gene) into Anabaena, the transgenic Anabaena produced no detectable isoprene. Next, we will optimize the IspS codon for Anabaena because the codon modified IspS gene worked in a cyanobacterium Synechocystis 6803 (Lindberg et al., 2010). Impacts: Our process will convert fermentation-derived CO2 into linalool or isoprene, increasing the profitability of biorefineries, with no additional feedstock cost. Engineering a cyanobacterium to directly convert CO2 to excreted end products, which bypasses the expensive, multiple unit operated biomass pathway currently used in cellulosic biofuels or algal oil production. Our model product linalool (C10H18O), a long-chain alcohol with an energy density of 40 mj/kg, heat of vaporization of 0.19 mj/kg, and octane of 102. These features also make linalool suitable for a drop-in biofuel. Linalool is a naturally-occurring terpene alcohol with many commercial applications, such as most frequently used as perfumed hygiene products and cleaning agents. Recently, it has been reported that 2 micro mole (2uM) of linalool is able to completely kill cancer cells (Usta etal., 2009).This will also make linalool as a potential anti-cancer drug. Assuming only half of the waste heat from a 100 MMGY corn ethanol plant can be captured, it would heat 1 million square ft (23 acres) of greenhouse space. This would be sufficient to produce up to 20,000 tons of linalool using our system, assuming only 50% CO2 fixation. This system will recycle water in the process, not use/generate hazardous/toxic substances, and reduce CO2 emissions by 2,000 tons for every million gallons of ethanol biomass ethanol produced. The low-impact environmental footprint will foster rural economic development, and provide an excellent ROI opportunity for corn and biomass-based ethanol facilities.
Publications
- No publications reported this period
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