Reducing carbon dioxide (CO2) emissions has been a hot research topic in recent years. The integration of microalgae cultivation using CO2 from power plants and factories has been introduced as an environmentally friendly approach. However, strains with high biomass productivity are required to achieve a sustainable integrated platform. Improving photosynthesis is critical to increase both biomass productivity and CO2 sequestration efficiency. The improvement of photosynthesis is often attained by enhancing the efficiency of enzymes that are involved in CO2 fixation, reducing the antenna size to avoid energy loss, extending the photosynthetically active radiation range to broaden the light utilization capacity, increasing CO2 assimilation by replacing the existing carbon fixation pathway with more efficient pathways and enzymes, and reducing the release of captured CO2. Implementation of these modifications is achievable via transformation and gene editing. The transformation of the new gene constructs into microalgae has been discussed as an extremely challenging task in the past decade. In recent studies, the digestion of the microalgae cell wall, as one of the main barriers of transformation, has been recommended as a promising approach. Moreover, the emergence of preassembled Cas9 protein-gRNA ribonucleoproteins that do not require vector constructs has been suggested as an efficient approach for gene editing. This review comprehensively describes the potential strategies that enhance microalgae CO2 fixation, provides insight into current limitations and gaps, and proposes future perspectives.

Recent progress in genetically modified microalgae for enhanced carbon dioxide sequestration

The capture of CO2 by carboxylases is key to sustainable biocatalysis and a carbon-neutral bio-economy, yet currently limited to few naturally existing enzymes. Here, we developed glycolyl-CoA carboxylase (GCC), a new-to-nature enzyme, by combining rational design, high-throughput microfluidics and microplate screens. During this process, GCC’s catalytic efficiency improved by three orders of magnitude to match the properties of natural CO2-fixing enzymes. We verified our active-site redesign with an atomic-resolution, 1.96-A cryo-electron microscopy structure and engineered two more enzymes that, together with GCC, form a carboxylation module for the conversion of glycolate (C2) to glycerate (C3). We demonstrate how this module can be interfaced with natural photorespiration, ethylene glycol conversion and synthetic CO2 fixation. Based on stoichiometrical calculations, GCC is predicted to increase the carbon efficiency of all of these processes by up to 150% while reducing their theoretical energy demand, showcasing how expanding the solution space of natural metabolism provides new opportunities for biotechnology and agriculture. Biological CO2 fixation is restricted to few enzymes and pathways, limiting its value in environmental protection and agricultural productivity. Now, a new-to-nature CO2-fixing enzyme allows CO2-dependent assimilation of glycolate in a designed pathway, and its use for different applications is demonstrated.

/pdf/a-new-to-nature-carboxylation-module-to-improve-natural-and-m6aqwmgksp.pdf

A new-to-nature carboxylation module to improve natural and synthetic CO2 fixation

Solar-driven coupling of water oxidation with CO2 reduction sustains life on our planet and is of high priority in contemporary energy research. Here, we report a photoelectrochemical tandem device that performs photocatalytic reduction of CO2 to formate. We employ a semi-artificial design, which wires a W-dependent formate dehydrogenase (FDH) cathode to a photoanode containing the photosynthetic water oxidation enzyme, Photosystem II, via a synthetic dye with complementary light absorption. From a biological perspective, the system achieves a metabolically inaccessible pathway of light-driven CO2 fixation to formate. From a synthetic point of view, it represents a proof-of-principle system utilizing precious-metal-free catalysts for selective CO2-to-formate conversion using water as an electron donor. This hybrid platform demonstrates the translatability and versatility of coupling abiotic and biotic components to create challenging models for solar fuel and chemical synthesis.

Photoreduction of CO2 with a Formate Dehydrogenase Driven by Photosystem II Using a Semi-artificial Z-Scheme Architecture.

Engineering Microorganisms for Enhanced CO2 Sequestration

CO2 sequestration engineering is an attractive strategy for achieving carbon- and energy-efficient bioproduction. However, the efficiency of heterotrophic CO2 sequestration is limited by bioproduct dependence and energy deficiency. Here, modular CO2 sequestration engineering was developed to produce target chemicals by integrating synthetic CO2 fixation and CO2 mitigation modules. A synthetic CO2 fixation pathway was designed, and then enhanced by light-driven reducing power using self-assembled cadmium sulfide nanoparticles. Next, a CO2 mitigation switch was designed, and then optimized by light-driven energy via proteorhodopsin. Finally, by integrating CO2 fixation and CO2 mitigation modules, the efficiency of CO2 sequestration was notably enhanced in Escherichia coli and the yields of l-malate and butyrate were increased to 1.48 and 0.79 mol/mol glucose, respectively, reaching theoretical yields. This CO2 sequestration system provides an efficient platform for channelling CO2 into value-added chemicals. Improving the efficiency of carbon yield in heterotrophic microorganisms is desired for biomanufacturing. Now, a product-independent and energy-efficient CO2 sequestration system that maximizes carbon conversion has been developed, as showcased by the production of chemicals reaching their theoretical yields.

Light-driven CO2 sequestration in Escherichia coli to achieve theoretical yield of chemicals

The Calvin-Benson-Bassham (CBB) cycle is presumably evolved for optimal synthesis of C3 sugars, but not for the production of C2 metabolite acetyl-CoA. The carbon loss in producing acetyl-CoA from decarboxylation of C3 sugar limits the maximum carbon yield of photosynthesis. Here we design a synthetic malyl-CoA-glycerate (MCG) pathway to augment the CBB cycle for efficient acetyl-CoA synthesis. This pathway converts a C3 metabolite to two acetyl-CoA by fixation of one additional CO2 equivalent, or assimilates glyoxylate, a photorespiration intermediate, to produce acetyl-CoA without net carbon loss. We first functionally demonstrate the design of the MCG pathway in vitro and in Escherichia coli. We then implement the pathway in a photosynthetic organism Synechococcus elongates PCC7942, and show that it increases the intracellular acetyl-CoA pool and enhances bicarbonate assimilation by roughly 2-fold. This work provides a strategy to improve carbon fixation efficiency in photosynthetic organisms.

/pdf/augmenting-the-calvin-benson-bassham-cycle-by-a-synthetic-35qy6ezj5j.pdf

Augmenting the Calvin-Benson-Bassham cycle by a synthetic malyl-CoA-glycerate carbon fixation pathway.

Cyanobacteria as a Host Organism

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Augmenting the Calvin-Benson-Bassham cycle by a synthetic malyl-CoA-glycerate carbon fixation pathway.

Cyanobacteria as a Host Organism