Engineering Bacteria to Mitigate Climate Change

By Ellie Fung

When considering climate change mitigation, images of a forest of wind turbines overlooking expansive fields of solar panels and sleek electric cars whirring past towering green buildings often come to mind. After all, grand challenges necessitate grand solutions. However, interest has been growing recently in the manipulation of microbial metabolic systems for the alleviation of climate change. Such biotechnical innovations have been spurred by advancements in synthetic biology – the design of new biological systems that do not exist naturally or modification of existing ones to perform non-native functions. 

With rapidly increasing atmospheric CO2, there has been much interest in CO2 reduction technologies. For one, carbon capture and sequestration involves the acquisition and transport of emitted CO2 to be stored in geological formations, such as in deep oceans or depleted oil reservoirs. However, this approach is associated with high capital and operating costs as well as large, potentially obtrusive infrastructure and risk of CO2 leakage. Contrastingly, manipulating microbes to capture CO2 may represent a safer and more cost-effective way to reduce CO2 (François, Lachaux & Morin, 2020). As a part of photosynthesis in autotrophic organisms, carbon fixation via the Calvin-Benson-Bassham (CBB) cycle is critical in maintaining the global carbon balance. Boosting this pathway in plants and algae may be a viable strategy to reduce atmospheric CO2, but growth rates of these organisms are much slower and genetic engineering is significantly more challenging compared to heterotrophic bacteria (Gong et al., 2015; Herz et al., 2017). Thus, there has been growing interest in genetically manipulating heterotrophic bacterial strains, such as E. coli, to autocatalytically synthesise sugars from inorganic CO2.  

Except for phosphoribulokinase (prk) and ribulous-1,5-bisphosphate cabosylase/oxygenase (Rubisco), the native metabolic enzymes in E. coli are able to catalyse CBB cycle reactions. The heterologous introduction of prk and Rubisco would theoretically enable a switch to an autotrophic lifestyle (Antonovsky et al., 2016). With this rationale, researchers have successfully introduced the CBB cycle in E. coli with the knockout of various enzymes involved in central metabolism and heterologous expression of prk and Rubisco (Antonovsky et al., 2016). To maintain sugar biosynthesis from CO2, adaptor laboratory evolution was also conducted on the engineered strains. This involved growing the strains in a xylose-limited chemostat with enriched CO2 to impose a strong selective pressure for bacteria to use CO2 instead of organic xylose in the CBB cycle. Consequently, metabolic flux was skewed towards the non-native autotrophic pathways instead of the heterotrophic pathways of which E. coli is adapted for, enabling biosynthesis of sugar purely from CO2. Though this demonstrated the potential of converting metabolic tropism in bacteria, only a “hemiautotrophic” metabolic status was achieved since the exogenous supplementation of an organic carbon source, such as pyruvate, was necessary to provide reducing power and additional metabolites (Antonovsky et al., 2016; Herz et al., 2017).

Similarly involving integration of heterologous gene expression, metabolic rewiring and laboratory evolution, recent breakthrough saw the development of a fully autotrophic E. coli strain. Here, formate dehydrogenase was introduced to generate NADH and ATP to power the CBB cycle from formate, which was provided in excess in the chemostat. Formate was selected as it is a byproduct of multiple processes, such as from fossil fuel combustion and renewable energy sources. True autotrophs were generated, where sugar biosynthesis required no organic carbon input given that formate supports E. coli growth assimilation into biomass. Though growth on formate produces net CO2 emissions as formate oxidisation to CO2 occurs at a quicker rate than CO2 assimilation into biomass in E. coli (Gleizer et al., 2019), this achievement nevertheless presents an exciting framework for reducing atmospheric carbon. In the future, careful assessment and planning must be undertaken before large-scale employment of these engineered bacteria to ensure natural ecosystems are not perturbed (DeLisi et al., 2020). Promisingly, this strain may serve as a model for the sustainable production of animal and human feed, biofuels and value-added chemicals. Providing metabolically engineered strains with formate generated by renewable carbon-negative resources may simultaneously enable atmospheric carbon capture and sustainable bioproduction (Gleizer et al., 2019; Yishai et al., 2016).

Along these lines, significant effort has been put into metabolically engineering bacteria to produce industrially important chemicals from low-cost renewable carbon resources. Notably, E. coli has been successfully engineered to produce 1,4-butanediol (BDO), a chemical made from oil and natural gas that is used in the manufacture of polymers. As BDO is not naturally synthesised by any known organism, there was no existing metabolic pathway to serve as a blueprint. Instead, computational genome-scale metabolic modelling and pathway prediction algorithm were used instead to identify groups of functionally related genes that participate in potential BDO production pathways. Out of 10,000 predicted pathways, one that involved known enzymes was selected to be introduced into E. coli, bypassing the need for additional protein engineering. The pathway was constructed in E. coli with knockout of central metabolic genes and optimisation of the citric acid cycle under anaerobic growth conditions, generating a strain that produced 18g L-1 BDO from glucose during batch fermentation (Nielsen, 2011; Yim et al., 2011). Such innovation may thus reduce reliance on fossil fuels for industrial chemical production, especially in lieu of populations and economies growing under the threat of climate change. 

Though BDO has since achieved production at commercial scales, few other compounds produced in this way have reached similarly high yields (Chubukov et al., 2016). This is in part due to the many challenges encountered at each stage of the bioengineering process, namely gene discovery, pathway construction, host metabolism engineering and process engineering, before implementation in bacterial strains can be successful. For instance, heterolologous gene expression does not necessarily mean that synthetic pathways function effectively in host strains. Multiple optimisation steps need to be undertaken to ensure suitable protein expression levels, alleviation of toxic non-native metabolites and diversion of metabolic flux to the desired pathway (Chubukov et al., 2016). Furthermore, systems that function within laboratories are often sensitive to slight changes or do not translate into industrial fermentation, especially since microbial metabolic processes are relatively inefficient (Nielsen, 2011). At present, the complexity of metabolic engineering means that it is considerably more labour-intensive and time-consuming than mechanical or civil engineering (Chubukov et al., 2016).

Despite its infancy, synthetic biology has already demonstrated significant potential as an effective strategy for climate change mitigation. In contrast to the utopian eco-city, perhaps the ideal way to alleviate this threat is by paying homage to the ubiquitous microorganisms and biological systems that too often goes unnoticed.

References:

Antonovsky, N., Gleizer, S., Noor, E., Zohar, Y., Herz, E., Barenholz, U., Zelcbuch, L., Amram, S., Wides, A., Tepper, N., Davidi, D., Bar-On, Y., Bareia, T., Wernick, D. G., Shani, I., Malitsky, S., Jona, G., Bar-Even, A. & Milo, R. (2016) Sugar Synthesis from CO2 in Escherichia coli. Cell. 166 (1), 115-125. Available from: doi: 10.1016/j.cell.2016.05.064. 

Chubukov, V., Mukhopadhyay, A., Petzold, C. J., Keasling, J. D. & Martín, H. G. (2016) Synthetic and systems biology for microbial production of commodity chemicals. Npj Systems Biology and Applications. 2 (1), 16009. Available from: doi: 10.1038/npjsba.2016.9. 

DeLisi, C., Patrinos, A., MacCracken, M., Drell, D., Annas, G., Arkin, A., Church, G., Cook-Deegan, R., Jacoby, H., Lidstrom, M., Melillo, J., Milo, R., Paustian, K., Reilly, J., Roberts, R. J., Segrè, D., Solomon, S., Woolf, D., Wullschleger, S. D. & Yang, X. (2020) The Role of Synthetic Biology in Atmospheric Greenhouse Gas Reduction: Prospects and Challenges. BioDesign Research. 2020 1016207. Available from: doi: 10.34133/2020/1016207. 

François, J. M., Lachaux, C. & Morin, N. (2020) Synthetic Biology Applied to Carbon Conservative and Carbon Dioxide Recycling Pathways. Frontiers in Bioengineering and Biotechnology. 7 446. Available from: https://www.frontiersin.org/article/10.3389/fbioe.2019.00446

Gleizer, S., Ben-Nissan, R., Bar-On, Y., Antonovsky, N., Noor, E., Zohar, Y., Jona, G., Krieger, E., Shamshoum, M., Bar-Even, A. & Milo, R. (2019) Conversion of Escherichia coli to Generate All Biomass Carbon from CO2. Cell. 179 (6), 1255-1263.e12. Available from: doi: 10.1016/j.cell.2019.11.009. 

Gong, F., Liu, G., Zhai, X., Zhou, J., Cai, Z. & Li, Y. (2015) Quantitative analysis of an engineered CO2-fixing Escherichia coli reveals great potential of heterotrophic CO2 fixation. Biotechnology for Biofuels. 8 86-1. eCollection 2015. Available from: doi: 10.1186/s13068-015-0268-1. 

Herz, E., Antonovsky, N., Bar-On, Y., Davidi, D., Gleizer, S., Prywes, N., Noda-Garcia, L., Lyn Frisch, K., Zohar, Y., Wernick, D. G., Savidor, A., Barenholz, U. & Milo, R. (2017) The genetic basis for the adaptation of E. coli to sugar synthesis from CO2. Nature Communications. 8 (1), 1705. Available from: doi: 10.1038/s41467-017-01835-3. 

Nielsen, L. K. (2011) From retrofitting to green field. Nature Chemical Biology. 7 (7), 408-409. Available from: doi: 10.1038/nchembio.601. 

Yim, H., Haselbeck, R., Niu, W., Pujol-Baxley, C., Burgard, A., Boldt, J., Khandurina, J., Trawick, J. D., Osterhout, R. E., Stephen, R., Estadilla, J., Teisan, S., Schreyer, H. B., Andrae, S., Yang, T. H., Lee, S. Y., Burk, M. J. & Van Dien, S. (2011) Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nature Chemical Biology. 7 (7), 445-452. Available from: doi: 10.1038/nchembio.580. 

Yishai, O., Lindner, S. N., Gonzalez de la Cruz, J., Tenenboim, H. & Bar-Even, A. (2016) The formate bio-economy. Current Opinion in Chemical Biology. 35 1-9. Available from: doi: S1367-5931(16)30096-5 [pii]. 

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