At-scale capture, storage, and utilization of greenhouse gasses (GHGs) by engineered organisms.

Engineering Biology for Climate & Sustainability

Biosequestration of Greenhouse Gases Goal:

At-scale capture, storage, and utilization of greenhouse gasses (GHGs) by engineered organisms.

Current State-of-the-Art

Removal of greenhouse gases – including carbon oxides, methane, nitrous oxide, and fluorinated gases – from the environment is one of the primary components to mitigating climate change. Using autotrophic organisms to capture GHGs, we can leverage the self-replication of biological organisms as a mechanism for continual capture, resulting in negative carbon emissions and a cleaner environment world wide. Biology is uniquely suited to address GHG capture, storage and utilization. It is likely that the first complex molecules to emerge on Earth were all synthesized from CO₂. ¹ and today several CO₂ fixation routes are known.²^,³^,⁴

Photoautotrophs (plants, algae, cyanobacteria) absorb sunlight and CO₂ to make biomass. Engineering biology could increase the efficiency of this process and create more capacity for CO₂ drawdown by using genetic editing tools to optimize key complexes, enzymes, and pathways involved in photosynthesis and carbon fixation. Advances in engineering biology, especially the emergence and widespread use of CRISPR, have led to a series of recent successes in engineering plants, though major research questions and challenges still remain.⁵ Extensive research efforts have been directed towards engineering RuBisCo – the enzyme responsible for catalyzing the first step of CO₂ uptake in carbon fixation and the most abundant protein on Earth turning over an approximate 400 gigatons of CO₂ per year – as a key target for improving plant photosynthesis efficiency to improve its catalytic efficiency.⁶ In addition to improving enzymatic pathways for CO₂ conversion, engineering photosynthetic organisms (especially plants) to more efficiently capture light and tolerate dynamic lighting conditions will help to achieve higher rates of CO₂ conversion.⁷ In addition to plants, photosynthetic organisms like cyanobacteria and algae are also valuable research targets for carbon capture. Importantly, cyanobacteria and algae contain carbon concentrating mechanisms (CCM) that make them more efficient at photosynthesis and carbon fixation than plants, and research is underway to embed CCMs into plants and other model organisms for carbon capture.⁸

In addition to these photoautotrophs that require light as a source of electrons, there is a wide range of chemoautotrophs capable of utilizing carbon oxides or methane.⁹^,¹⁰ Efforts are underway to develop tools to efficiently engineer chemoautotrophic organisms including acetogens, hydrogenogens, or methanotrophs or even transfer into model organisms like E.coli or yeast.¹¹^, ¹² This includes enhancing the seven known CO₂ fixation pathways with new-to-nature reactions or designing synthetic or de novo CO₂ fixation pathways. Researchers have aimed to circumvent the challenges posed by endogenous carbon fixation by focusing on designing synthetic metabolic pathways¹³^, ¹⁴ and identifying key enzymes other than RuBisCo that are critical for carbon fixation, such as carboxylation via 6-phosphogluconate dehydrogenase.¹⁵^, ¹⁶ There is also work underway to rewire CO₂ fixation pathways¹⁷^, ¹⁸ or transplant engineered fixation pathways into (new) microbial chassis and engineering in vitro CO₂ fixation in cell-free systems.¹⁹ Key challenges include that there are still gaps in our understanding of CO₂ fixation pathways²⁰^, ²¹^, ²² and many pathways such as the Wood-Ljundahl pathway which is considered to be the most energy efficient CO₂ fixation pathway²³^, ²⁴^, ²⁵ are complex and require a network of hundreds of genes involved for chemoautotrophic growth and associated energy conservation.²⁶

Most chemoautotrophs convert carbon oxides or methane into cellular biomass or simple molecules such as acetate (which are intermediates for other organisms in the global carbon cycle).²⁷^, ²⁸ Engineered organisms and biobased systems could upgrade intermediates like acetate,²⁹ or capture and convert carbon oxides directly, into more complex, value-added commodities.³⁰^, ³¹^, ³² Because many photosynthetic and chemoautotrophic organisms convert CO₂ into biomass through carbon fixation, essentially turning gaseous CO₂ into solid carbon, they conveniently achieve carbon capture and storage at the same time, enabling carbon negative manufacturing.³³ For instance, bacteria could be engineered to convert carbon oxides into precursors for acrylic glass,³⁴ bioplastics,³⁵ or solid compounds like calcium carbonate,³⁶ which could keep captured CO₂ sequestered for tens to hundreds of years.³⁷ Such approaches could further help mitigate the risk of uncontrolled release from carbon capture and storage. Already, ethanol production from carbon-monoxide rich industrial off-gases with native chemoautotrophs is carried out at commercial scale by companies like LanzaTech. Charm Industrial, a carbon tech startup, aims to “permanently put CO₂ back underground” by making bio-oil from the pyrolysis of waste biomass and injecting the oil into deep geological formations. Recent research has demonstrated the biosynthesis of starch from CO₂ in cell-free systems,³⁸ the production of cotton-alternative cellulose from CO₂,³⁹ and the production of value-added chemicals in co-cultured microbial consortium.⁴⁰ Similarly, a range of chemical production from methane has been described in engineered methanotrophs.⁴¹^, ⁴²^, ⁴³^, ⁴⁴ In addition to CO₂ and methane conversion, capturing and conversion carbon oxide containing off-gases from heavy industry (e.g., steel, ferroalloy) or syngas from gasification of various solid wastes via microbial gas fermentation into a range of chemicals has been demonstrated⁴⁵ and a recent study demonstrated production of platform chemicals acetone and isopropanol at high rates in an industrial pilot.⁴⁶ Further, macroalgae could sequester nitrates and phosphates, followed by harvesting and use as low/negative-carbon fertilizers. Where no concentrated CO₂ or methane stream is available as required for many conversion or storage technologies, biology may also provide an opportunity to increase the concentration of gases, as an alternative to current direct air capture (DAC) methods.⁴⁷

The processes described above could be used to store and utilize GHGs captured at emission sources. Concentrated streams, such as emissions from power plants, are easier to mitigate than diluted sources, such as diffuse GHGs in the atmosphere. While engineered organisms are currently tested in lab settings using controlled amounts of CO₂ or methane as input, we still need to develop engineering capabilities to enable the biosequestration of environmental and diffuse carbon at an industrial scale. Improving gas fermentation technology will be key to accomplishing this.⁴⁸^, ⁴⁹ These capabilities will be important stepping stones towards enabling organisms to capture different types of GHGs from concentrated streams and ambient air and convert captured GHG molecules into value-added products.

Breakthrough Capabilities & Milestones

Improve CO₂uptake by engineering more efficient photosynthetic organisms (plants, algae, cyanobacteria).

Engineer plants for optimized light collection and more efficient use of captured light for photosynthesis.

Bottleneck/Challenge: Chlorophylls have evolved to only absorb light in the wavelength range of 400nm to 700nm.

Potential Solution: Engineer and introduce into plants alternative chlorophylls with expanded absorption spectrum, such as by enabling the expression of bacteriochlorophylls, which have absorption maxima in the far-red region.

Bottleneck/Challenge: Light harvesting complexes (antennae proteins) trap more light than can be used for photochemistry and block leaves in lower layers from accessing more light.

Potential Solution: Engineer photosystems to have a reduced number of antennae or smaller antennae.

Bottleneck/Challenge: Photoprotective mechanisms, such as non-photochemical quenching (NPQ), protect the plant from excess light, but decrease the overall photosynthetic efficiency under high-light conditions.

Potential Solution: Introduce genes into plants to accelerate the relaxation rate of photoprotection and NPQ.

Potential Solution: Incorporate genes (and engineer new circuits and regulatory networks, as necessary) that enable plants to quickly adapt to fluctuating light conditions and turn off photoprotection, so excess light is used towards photosynthesis instead of being dissipated as heat.

Engineer pathways and enzymes in photosynthetic organisms to increase the rate and efficiency of carbon fixation.

Bottleneck/Challenge: Genetic engineering tools developed in model organisms are often ineffective or inefficient in photosynthetic organisms.

Potential Solution: Develop metabolic models and genetic engineering tools for photosynthetic organisms.

Potential Solution: Bioprospect for new organisms to expand basic understanding of the molecular biology of photosynthetic microbes.

Bottleneck/Challenge: : RuBisCO is a large complex made of multiple subunits that require an array of chaperones for folding and assembling and that are sensitive to inhibition by sugar-phosphate ligands;⁵⁰ engineering its catalytic biochemistry currently requires non-ideal tradeoffs.

Potential Solution: Develop better understanding of RuBisCo components, such as via high-throughput microfluidic enzyme kinetics, to enable editing multiple aspects simultaneously.⁵¹^, ⁵²

Potential Solution: High-throughput characterization of the biodiversity of RuBisCos across photosynthetic organisms to identify those that have fewer subunits, simpler folding kinetics, and high efficiency; engineer existing elements of those systems into photosynthetic organisms that are or can be grown at scale.

Bottleneck/Challenge: RuBisCo has an error rate of more than 20% resulting in toxic 2-phosphoglycolate, with engineering efforts to improve has been challenging; 2-Phosphoglycolate salvage is an energetically expensive and wasteful process, losing a carbon in the form of CO₂.⁵³^, ⁵⁴

Potential Solution: Modifying 2-Phosphoglycolate salvage to improve carbon efficiency.

Potential Solution: Engineer and transform plants with more efficient RuBisCO.⁵⁵

Bottleneck/Challenge: Challenges remain in expressing prokaryotic carbon concentrating mechanisms (CCM) in eukaryotic cells.

Potential Solution: Engineer fully reconstructed heterologous CCMs to enable successful expression of CCM in plants and model organisms.

Potential Solution: Engineer microbes to grow using captured carbon as substrates.

Develop scalable carbon capturing platforms enabled by engineered green algae and cyanobacteria.

Bottleneck/Challenge: For algae farms coupled to carbon emitters (e.g., power plants), there is more CO₂ emitted than the algae farm could fully capture and utilize.

Potential Solution: Select and engineer algal strains with high CO₂ uptake rates and/or carbon concentrating mechanisms.

Potential Solution: Engineer hydrogenases to increase carbon utilization in green algae (e.g., hydrogenases not inhibited by carbon monoxide).

Bottleneck/Challenge: Current photo-bioreactor design is insufficient to optimize carbon capture and bioproduction.

Potential Solution: Construct low-cost open bioreactors (e.g., ponds, photobioreactors, or gas fermentors) with organism-tailored geometries, flow rates, and media compositions.

Combine and rewire native CO₂ fixation pathways (e.g., C3 and C4 pathways) and engineer organisms capable of utilizing multiple carbon fixation pathways (see for example Moreno-Villena et al., 2022*).

Bottleneck/Challenge: Engineering C3 plants for C4 carbon fixation requires control of the precise spatial expression of many genes between mesophyll and bundle sheath cells, which would be challenging and very time intensive to engineer in plants.⁵⁶

Potential Solution: Improved techniques for transforming plants with multiple genes and/or pathways under precise spatial control.

Bottleneck/Challenge: Engineering many elements of a pathway is challenging; the expression and function of each element may need to be optimized, e.g., to avoid the production of undesirable intermediates or finetune pathway regulatory mechanisms.⁵⁷

Potential Solution: Optimize rapid in vitro and in vivo pathway characterization.

*Moreno-Villena, J. J., Zhou, H., Gilman, I. S., Tausta, S. L., Cheung, C. Y. M., & Edwards, E. J. (2022). Spatial resolution of an integrated C4+CAM photosynthetic metabolism. Science Advances, 8(31), eabn2349. View Publication.

Enable efficient carbon capture by engineered chemoautotrophs.

Map and identify parts in CO₂ fixation pathways to increase the efficiency of carbon fixation in chemoautotrophic organisms.

Bottleneck/Challenge: Identity and understanding of the most rate-limiting step to CO₂ sequestration in chemoautotrophic model organisms and the missing energy-coupling sites and interaction in native carbon fixation pathways (e.g., Wood-Ljungdahl pathway).

Potential Solution: Understand the role of all genes involved in carbon fixation in chemoautotrophic organisms through omics approaches, enzyme studies, mutagenesis or knockout experiments to identify the rate-limiting step and missing links.

Potential Solution: Map and understand the flux and bioenergetic links between carbon, nitrogen, phosphorus, sulfur metabolism in chemoautotrophs.

Bottleneck/Challenge: Knowledge of how changes in enzyme expression levels affect function in C1 pathways.

Potential Solution: Map protein-protein interactions, characterize transcription factors and multienzyme complexes and their dynamics, and identify metabolic substrate channeling between relevant enzymes.

Engineer complexes and metabolic pathways in chemoautotrophs to improve carbon fixation.

Bottleneck/Challenge: Enzymes and cofactors optimized for recycling and energetics.

Potential Solution: Improve the efficiency of major CO₂ fixation or methane oxidizing enzymes.

Potential Solution: Discover or design new enzymes that are more efficient at capturing CO₂ or converting methane.

Potential Solution: Develop orthologous co-factors.

Bottleneck/Challenge: Limited molecular and genetic toolkits for domesticated chemoautotrophs.

Potential Solution: Develop broader toolsets (e.g., genome engineering, enzyme engineering, and cell-free systems) and high-throughput workflows for engineering chemoautotrophs, such as Thermotoga neapolitana, Cupriavidus necator, Clostridia species, and methanoarchaea.

Potential Solution: Develop high-throughput screening capabilities to reduce strain development cycle times.

Bottleneck/Challenge: High-throughput cultivation and product screening in context flammable and/or toxic gaseous substrates such as carbon oxides and methane.

Potential Solution: Develop new plate based or microfluidics based screening workflows that facilitate growth on gaseous substrates, while retaining or direct measuring of product concentrations.

Potential Solution: Develop analytics and sensor tools for dissolved concentrations of carbon oxide and methane gasses in screening assays.

Demonstrate use of engineered chemoautotrophs to capture more CO₂ in the context of environmental or industrial processes.

Bottleneck/Challenge: Air and many other potential industrial streams (e.g., cement plants, landfills) have low CO₂ or methane concentrations requiring expensive steps for gas concentration or compression.

Potential Solution: Engineer organisms for effective conversion at low or atmospheric CO₂ or methane concentrations.

Bottleneck/Challenge: Effective biocontainment strategies for deployed organisms.

Potential Solution: Develop low-cost methods to employ bio-orthogonal biochemistry.

Potential Solution: Develop risk analysis frameworks to define risk benchmarks.

Enable organisms to utilize captured carbon to produce value-added chemicals and materials.

Engineer organisms to convert CO₂, methane, or other C1 sources and intermediates (incuding methanol, formate, acetate) into value-added compounds.

Bottleneck/Challenge: Optimal electro-biochemical routes for carbon conversion into value added compounds are not known.

Potential Solution: Design electro-biochemical routes for minimizing the loss of carbon through metabolism or to directly sequestering carbon for bioconversion into value-added compounds.⁵⁸

Potential Solution: Develop approaches to evolve promising chemolithoautotrophic organisms to increase yield of desired products.

Bottleneck/Challenge: Lack of platforms for genome-wide engineering of non-model chemoautotrophs with metabolic and physiological capabilities needed for optimized carbon conversion.

Potential Solution: Develop new genome scale modeling and engineering tools for rapidly generating and implementing carbon-optimized designs.

Potential Solution: Develop machine learning algorithms, artificial intelligence tools, cell-free systems, and multi-omics workflows to enable faster data-driven DBTL cycles in non-model microbes.

Bottleneck/Challenge: While acetate is a universal carbon source for many microbes (including model organisms such as yeast or E. coli) that have been engineered to produce value-added chemicals, the current process releases CO₂.⁵⁹

Potential Solution: Chemoautotrophs are capable of producing acetate from CO₂ at high rates;⁶⁰ adapt efficient production strains for using acetate instead of sugars as substrate for value-added products and develop co-culture or coupled processes.

Optimize the bio-utilization of CO₂ and methane emitted from point sources.

Bottleneck/Challenge: High gas mass transfer is required; gases like methane, carbon monoxide or hydrogen are poorly soluble.

Potential Solution: Develop energy-efficient systems for harvesting products made by microbes grown in large-scale bioreactors.

Bottleneck/Challenge: Waste gas streams contain compounds that inhibit the activities of microbes and enzymes.

Potential Solution: Engineer and select microbes to tolerate different sources of greenhouse gas and metabolic byproducts.

Potential Solution: Improve enzymatic activity, stability, and reusability for converting CO₂ into chemicals.

Improve gas fermentation technologies.

Combine and rewire native carbon utilization pathways and engineer organisms capable of using multiple carbon metabolism pathways.

Bottleneck/Challenge: Flexible chassis organisms suitable for industrial scale cultivation.

Potential Solution: Engineer reversible flux-based CO₂ fixation, H2 production and methanogenesis/methanotrophy in, for example, Methanosarcinales.⁶¹

Potential Solution: Engineer consortia that can capture and utilize the full carbon life-cycle in a circular manner.

Enable carbon capture and utilization by enzymes or cell-free systems.

Develop efficient enzymes for concentrating carbon from the atmosphere.

Bottleneck/Challenge: Current methods for direct air capture (DAC) technologies to concentrate CO₂ from air are expensive.⁶²

Potential Solution: Enzymes like carbonic anhydrase (CA) can facilitate the dissolution of atmospheric CO₂ but require improvement in efficiency, stability, and inexpensive ways to release CO₂ for downstream processes.

Develop scalable cell-free systems as platforms for carbon capture and bioconversion.

Develop efficient and scalable cell-free systems capable of utilizing methane, formate, or CO₂ to produce commodity fuels and chemicals.

Bottleneck/Challenge: Modular capabilities within cell-free systems to produce high-value products.

Potential Solution: Engineer efficient multienzyme (plug-and-play, step-wise) cascade systems to convert CO₂.

Bottleneck/Challenge: Many methane-capturing enzymes, such as methane monooxygenase (MMO), are membrane-associated and thus more challenging to develop for cell-free technologies.

Potential Solution:Advance methods for creating vesicles or lipid discs enriched with functionally active MMOs that can be used to supplement cell-free systems with membrane-associated activities.

Develop self-contained and/or standalone cell-free CO₂ fixation systems for bio-enabled artificial photosynthesis.

Develop new platform tools for multienzyme immobilization in cell-free systems.

Footnotes

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