Background - The Climate Crisis
Carbon dioxide concentration levels in the atmosphere are higher now than at any other time in human history, and they are only projected to rise due to human activities.1 Carbon dioxide is a greenhouse gas, which plays a crucial role in regulating the surface temperature of the planet.1 As CO2 accumulates in the atmosphere, it leads to a net increase in average global surface temperatures.1
We are already beginning to experience the effects of climate change due to global warming - from more extreme rainfall, droughts, cyclones, and wildfires, to the extinction of numerous plant and animal species.2 The globe has already warmed by over 1°C above pre-industrial levels.1 In order to limit global warming to 1.5°C above pre-industrial levels, greenhouse gas emissions must fall by 45% by 2030, and we must achieve net-zero emissions by 2050.2
Problem - Petrochemical Synthesis
A carbon feedstock is a source of carbon that can be used as a raw material for manufacturing. Most carbon-based industrial chemicals rely on fossil fuels, like coal and crude oil, as raw material for their manufacture.3 However, the use of coal and oil for the production of these chemicals emits large amounts of carbon dioxide and other greenhouse gases that warm the atmosphere and oceans. The chemical and petrochemical industry is the third-largest source of industrial CO2 emissions, generating 1.5 gigatonnes of CO2 annually and accounting for 18% of the direct industrial CO2 emissions.3
A sustainable alternative to fossil fuel-based carbon feedstocks is to make use of biomass as a carbon feedstock. Heterotrophic bacteria can be engineered to consume carbohydrates from biomass and convert them into industrially important metabolites. However, relying on plant-based biomass like corn and sugarcane as raw material for industrial-scale chemical synthesis could compete with food production and advance environmental degradation4,5. Plant cultivation is often resource-intensive, requiring a lot of land, water, and fertilizers.6
A truly sustainable carbon source would need to be far more efficient.
A way forward - Photosynthesis
Cyanobacteria are photosynthetic prokaryotes that are far more efficient than plants at using sunlight to fix atmospheric CO2 into biomass; they can convert as much as 9% of solar energy received into biomass compared with only 0.5 - 3% for higher plants.7 They also require a fraction of the space and resources needed to cultivate plants.7 Cyanobacteria are more amenable to genetic manipulation than eukaryotic algae or plants8 and can be grown conveniently in a photobioreactor, on unproductive land, wastewater, and flue gas.8
Our solution - SynBactory
We have designed a sustainable biomanufacturing method to synthesize biobutanol from light and carbon dioxide, using a co-culture of two bacteria, Synechococcus elongatus UTEX 2973 and E. coli.
Our solution would minimize carbon emissions by not only capturing existing carbon emissions present in the atmosphere but will also prevent new emissions from being generated at the source by reducing the current reliance on carbon-emitting petrochemical feedstocks.
S. elongatus UTEX 2973 is a fast-growing strain of cyanobacteria, which accumulates sucrose intracellularly on exposure to salt stress.9 This sucrose is obtained from atmospheric carbon dioxide fixed by the bacterium during photosynthesis. When engineered to express a non-native sucrose transporter, cscB, the cyanobacterium can continually secrete the sucrose it generates.10 S. elongatus UTEX 2973 can secrete nearly 90% of its absorbed carbon as sucrose, as opposed to just 15% in sugarcane.10,9 This secreted sucrose can function as a renewable carbon feedstock for chemical synthesis via E. coli.
E. coli can be made to consume the sucrose generated by the cyanobacteria as its sole carbon source through the heterologous expression of the cscABK gene cluster, which facilitates the import and breakdown of sucrose.11 With an appropriate set of modifications, E. coli can be made to produce a desired metabolic product.
The system is inherently modular, in that one E. coli strain engineered to produce a particular metabolite, can be swapped with another microbial strain engineered to produce a different product.
As a proof of concept, we aimed to produce butanol through our co-culture by making use of the pre-engineered butanol producing E. coli KJK01 strain12 that we received from Dr. Syed Shams Yazdani at ICGEB, New Delhi, and a pre-engineered cscB expressing S. elongatus strain9 that we received from Dr. Himadri Pakrasi at Washington University in St. Louis.
We also designed in silico models to probe the stability and dynamics of the co-culture system to identify optimal conditions for the production of butanol. We used genome-scale metabolic models to identify key gene deletion and overexpression targets to improve butanol and sucrose yields. Furthermore, we designed constructs to characterize the strengths of native promoters to boost sucrose secretion in cyanobacteria.
Why a co-culture?
A co-culture is a cell cultivation setup in which two or more different populations of cells are grown with some degree of contact between them.
Our S. elongatus and E. coli co-culture facilitates a single pot, direct conversion of CO2 into the desired final product. A co-culture is more cost-effective and less wasteful since the requirements to recover and transport sucrose are eliminated because the E. coli would consume the sucrose directly.
A co-culture is mutually beneficial to both species; E. coli relies on S. elongatus for sucrose, and also benefits from the oxygen released by S. elongatus during photosynthesis. S. elongatus has been shown to grow better in the presence of E. coli, which are suspected of relieving oxidative stress by quenching reactive oxygen species.13
The primary use of biobutanol is as a fuel in internal combustion engines. Although ethanol and bio-diesel are preferred biofuels, it is expected that biobutanol will gain prominence in the next 5-10 years.18This is owing to biobutanol’s higher energy content, low volatility and corrosiveness, and its higher compatibility with existing vehicle and oil industries (it is a drop-in replacement to petroleum-based butanol)19.
Increasing urbanization globally has led to a rise in the demand for butanol-based products for the purpose of infrastructure and construction. It is also heavily used for the manufacturing of coatings for several end-user industries and is an essential intermediate in the production of butyl acrylate and adhesives. Its ability to be used as an additive has also increased demand in the pharmaceutical industry19.
The production mechanism primarily follows the process of Acetone–butanol–ethanol (ABE) fermentation, wherein obligate anaerobic bacteria (usually from the Clostridia class) ferment sugars in the feedstock to produce solvents. However the majority of the global bio-butanol production is from sugarcane and starch, and the remaining is from sugar beet, wheat, cellulosic sugars, and sweet sorghum juice. However, the tight supply of sugarcane and corn feedstock in the past few years, owing to their primary use in food consumption, has led to uncertainty in their availability for biobutanol production19.
Fluctuating raw material cost and availability pose tremendous challenges in the sustained large-scale production of biobutanol. A different carbon source as a feedstock is a possible solution that will alleviate the limitations of current production methods.
Sustainable Development Goals
The UN has proposed a bold new vision of sustainable development - an agenda to transform all nations of the world into peaceful, resilient, and prosperous societies for all humans20. At the core of the agenda are the seventeen sustainable development goals, or SDGs, including the eradication of poverty and hunger, the assurance of good health and living, the guarantee of a clean environment, action against climate change, sustainable human settlements, and responsible consumption. We hope that our project may work in consonance with these goals and be a small step towards goals 7 (“Affordable and Clean Energy”), 12 (“Responsible Consumption and Production”), and 13 (“Climate Action”).
A carbon-neutral route to produce chemicals and petrochemicals that annually account for 18% of the direct industrial CO2 emissions.3 would thus advance goals 12 and 13. In the case of products intended to be used as fuels - such as butanol - this would provide a form of clean energy as described in goal 7. The need for such an alternative gave birth to SynBactory - an application of synthetic biology and biomanufacturing to the greatest crisis of today’s age.
During our initial brainstorming sessions for a project idea, we created a rubric to decide how to pick a project. We all agreed that we wanted it to help solve a pressing, real-world problem and were heavily guided by the United Nations Sustainable Development Goals in our quest.
We explored ideas such as extracting metals from e-waste, engineering bacteria to ameliorate coral bleaching, implementing a machine learning algorithm in populations of E. coli, making biosurfactants, using RNA interference to fight plant diseases, amongst many others.
As we went about exploring wikis and finding pressing problems, we came upon the IPCC 2018 Special Report on Global Warming of 1.5 °C (SR15).2 The climate crisis and its far-reaching implications gripped us. At about the same time, we came across previous work done on cyanobacteria and discovered its excellent photosynthetic efficiency, robustness, and amenability to manipulation. We found the 2018 Stony Brooke University iGEM team's work on sucrose synthesis from S. elongatus, which helped us develop our idea of using secreted cyanobacterial sucrose to synthesize chemicals. We first decided to try and produce succinic acid from sucrose in E. coli by engineering it to consume sucrose and to redirect its carbon flux towards the accumulation of succinic acid, an intermediary step in the TCA cycle.14 Succinic acid is a platform chemical and an essential raw material for manufacturing polybutylene succinate (PBS), a biodegradable bioplastic.15,16
We also discovered the fast-growing strain of S. elongatus UTEX 2973, which had a significantly higher doubling time (comparable to yeast at optimal conditions) and produced the highest sucrose yields.9,17
However, with the devastating second wave of the COVID-19 pandemic that hit India in early April our hopes of returning to the lab were delayed significantly - particularly as our institute is located in the state of Maharashtra, which was hit extremely hard. We no longer had the time to engineer S. elongatus and E. coli from scratch, and given the number of modifications we would need to make in E.coli to produce succinic acid, we were looking to switch to another metabolite as a proof of concept of our co-culture system.
We were fortunate to receive help from two labs in providing us with pre-engineered strains to help us fast-forward to performing assays and measurements. Dr. Yazdani from ICGEB provided us with his butanol-producing KJK01 strain of E. coli, and Prof. Pakrasi provided us with his sucrose-exporting strains of S. elongatus UTEX 2973.
After months of anxious waiting, we finally managed to get back to campus and to our labs by mid-August when the second wave began to wane. We could finally work together in person instead of interacting over Discord or Google Meet from different parts of the country. Having been in the lab for just short of two months, we picked up a tremendous amount of lab skills and experience, even if we didn't obtain all the results we hoped for.
Cyanobacteria are challenging chasses to work with, and just a handful of labs across India specialise in their use. We were lucky to find our mentors, Prem Pritam and Virmal Jain, from IIT Bombay, who have experience working with S. elongatus and guided us remotely over the phone. Since our institute has no equipment and material designed to cultivate cyanobacteria, we had to do quite a bit of jugaad* to get around our constraints. We had to drive down to Mumbai to borrow some emergency BG-11 medium from Prem in IIT Bombay, use rubber bands to fasten our flasks to the incubator shakers due to a lack of appropriately sized holders, order a custom-made regulatable light source for the incubator from a vendor known to our mentors, and add bicarbonate to the medium as a substitute for a regulated carbon dioxide supply, which our incubator could not support. We have described some of the difficulties with working cyanobacteria in our document titled Cyanobacteria: Tips and Tricks that we have submitted as a contribution.
Being located in India poses its own challenges for research in synthetic biology Shipments of strains, plasmids, reagents, and DNA from Europe and America can take up to two or more months to arrive, which greatly affects the amount of lab work that can be done during the iGEM cycle. With the pandemic, these delays are often longer than expected. We are still waiting for some of our enzymes and plasmids to arrive.
In our case, the first shipment of S. elongatus UTEX 2973 strains from Prof. Pakrasi's lab in St. Louis, had gotten caught up at customs due to some errors in paperwork. When the strains did eventually arrive, the plates they were streaked on were damaged, and the agar had fallen off. We were unable to revive them. The Pakrasi lab generously offered to send us the strains once again; they arrived in perfect condition on 23rd August.
There are a number of constraints posed by this chassis, including:
- Much higher doubling times than E. coli
- Sensitivity to medium composition, light intensity, and carbon dioxide concentrations
- Density-dependent growth - cyanobacterial biomass cannot be used to inoculate large volumes of medium as they cannot grow in overly dilute conditions
- Specific requirements for the ratio between the surface area of the medium-air interface and volume of the flask to minimize evaporation
- Propensity to aggregate and/or enter stasis, slowing growth and making it harder to measure growth under OD.
The aforementioned constraints make it necessary to passage inocula of cyanobacterial periodically from low to high volumes over 3-4 days, before one can actually begin any work on them - whether it is plating, preparing stocks for cryopreservation, or performing growth assays. This is why most of our time in the lab went into the same, preventing us from further engineering and experimenting with the strains.
*Jugaad is a Hindi word that has no accurate translation to English but roughly translates to a quick, creative, non-conventional hack or fix to unforeseen problems.
- IPCC, 2021: Summary for Policymakers. In: Climate Change 2021: The Physical Science Basis. Contribution of Working Group I to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change [MassonDelmotte, V., P. Zhai, A. Pirani, S.L. Connors, C. Péan, S. Berger, N. Caud, Y. Chen, L. Goldfarb, M.I. Gomis, M. Huang, K. Leitzell, E. Lonnoy, J.B.R. Matthews, T.K. Maycock, T. Waterfield, O. Yelekçi, R. Yu, and B. Zhou (eds.)]. Cambridge University Press. In Press.
- IPCC, 2018: Summary for Policymakers. In: Global warming of 1.5°C. An IPCC Special Report on the impacts of global warming of 1.5°C above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, sustainable development, and efforts to eradicate poverty [V. Masson-Delmotte, P. Zhai, H. O. Pörtner, D. Roberts, J. Skea, P. R. Shukla, A. Pirani, W. Moufouma-Okia, C. Péan, R. Pidcock, S. Connors, J. B. R. Matthews, Y. Chen, X. Zhou, M. I. Gomis, E. Lonnoy, T. Maycock, M. Tignor, T. Waterfield (eds.)]. World Meteorological Organization, Geneva, Switzerland, 32 pp https://report.ipcc.ch/sr15/pdf/sr15_spm_final.pdf
- IEA (2018), The Future of Petrochemicals, IEA, Paris https://www.iea.org/reports/the-future-of-petrochemicals
- Gressel, Jonathan. (2008). Transgenics are imperative for biofuel crops. Plant Science. 174. 246-263.http://dx.doi.org/10.1016/j.plantsci.2007.11.009
- Furtado, A., Lupoi, J.S., Hoang, N.V., Healey, A., Singh, S., Simmons, B.A. and Henry, īR.J. (2014), Modifying plants for biofuel and biomaterial production. Plant Biotechnol J, 12: 1246-1258.https://doi.org/10.1111/pbi.12300
- Pimentel, D. Ethanol Fuels: Energy Balance, Economics, and Environmental Impacts Are Negative. Natural Resources Research 12, 127-134 (2003). https://doi.org/10.1023/A:1024214812527
- Dismukes, G. C., Carrieri, D., Bennette, N., Ananyev, G. M., & Posewitz, M. C. (2008). Aquatic phototrophs: efficient alternatives to land-based crops for biofuels. Current opinion in biotechnology, 19(3), 235-240.https://doi.org/10.1016/j.copbio.2008.05.007
- Knoot, C. J., Ungerer, J., Wangikar, P. P., & Pakrasi, H. B. (2018). Cyanobacteria: promising biocatalysts for sustainable chemical production. Journal of Biological Chemistry, 293(14), 5044-5052.https://doi.org/10.1074/jbc.R117.815886
- Lin, PC., Zhang, F. & Pakrasi, H.B. Enhanced production of sucrose in the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Sci Rep 10, 390 (2020). https://doi.org/10.1038/s41598-019-57319-5
- Ducat, D. C., Avelar-Rivas, J. A., Way, J. C., & Silver, P. A. (2012). Rerouting carbon flux to enhance photosynthetic productivity. Applied and environmental microbiology, 78(8), 2660-2668. https://doi.org/10.1128/AEM.07901-11
- Mohamed, E.T., Mundhada, H., Landberg, J. et al. Generation of an E. coli platform strain for improved sucrose utilization using adaptive laboratory evolution. Microb Cell Fact 18, 116 (2019).https://doi.org/10.1186/s12934-019-1165-2
- Ali Samy Abdelaal, Kamran Jawed, Syed Shams Yazdani, CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium, Journal of Industrial Microbiology and Biotechnology, Volume 46, Issue 7, 1 July 2019, Pages 965-975, https://doi.org/10.1007/s10295-019-02180-8
- Zhang, L., Chen, L., Diao, J. et al. Construction and analysis of an artificial consortium based on the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce the platform chemical 3-hydroxypropionic acid from CO2. Biotechnol Biofuels 13, 82 (2020).https://doi.org/10.1186/s13068-020-01720-0
- National Center for Biotechnology Information (2021). PubChem Compound Summary for CID 1110, Succinic acid. Retrieved October 17, 2021, from https://pubchem.ncbi.nlm.nih.gov/compound/Succinic-acid
- Ke-Ke Cheng, Gen-Yu Wang, Jing Zeng, Jian-An Zhang, "Improved Succinate Production by Metabolic Engineering", BioMed Research International, vol. 2013, Article ID 538790, 12 pages, 2013. https://doi.org/10.1155/2013/538790
- Sengupta, S., Jaiswal, D., Sengupta, A. et al. Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid. Biotechnol Biofuels 13, 89 (2020). https://doi.org/10.1186/s13068-020-01727-7
- Yu, J., Liberton, M., Cliften, P. et al. Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2. Sci Rep 5, 8132 (2015). https://doi.org/10.1038/srep08132
- n-Butanol Market by Application (Butyl Acrylate, Butyl Acetate, Glycol Ethers, Direct Solvents, Plasticizers), and Region (APAC, North America, Europe, Middle East & Africa, South America) - Global Forecast to 2025; marketsandmarkets.com Report Code CH 1543
- Bio-butanol Market Size, Share & Trends Analysis Report By Application, By Region (North America, Europe, Asia Pacific, RoW), And Segment Forecasts, 2016 - 2022; grandviewresearch.com Report ID: 978-1-68038-523-6