Microbial Carbon Capture in Single-Molecule Systems Using Genetically Engineered Diatoms

Engineering Diatom Proteins to Enhance CO₂ Sequestration Rates at the Molecular Level

The urgency of climate change has spurred scientists to explore unconventional solutions for carbon capture. Among the most promising avenues is the genetic engineering of diatoms—microscopic algae responsible for nearly 20% of global carbon fixation—to optimize their CO₂-sequestering proteins at the single-molecule level.

The Molecular Machinery of Diatom Carbon Capture

Diatoms possess a sophisticated carbon-concentrating mechanism (CCM) that enables them to thrive in low-CO₂ environments. At its core are three key protein components:

• Carbonic anhydrases (CAs): Zinc metalloenzymes that catalyze the interconversion between CO₂ and bicarbonate (HCO₃^-)
• Rubisco enzymes: Ribulose-1,5-bisphosphate carboxylase/oxygenase that fixes CO₂ in the Calvin cycle
• Bicarbonate transporters: Membrane proteins that actively concentrate HCO₃^- within the cell

Protein Engineering Strategies for Enhanced Performance

Recent advances in structural biology and directed evolution have enabled precise modifications to these molecular machines:

Carbonic Anhydrase Optimization

The α-CA isoform from Thalassiosira weissflogii has become a prime target for engineering due to its exceptional turnover rate (~10⁶ reactions per second). Researchers have successfully:

• Increased zinc-binding affinity through histidine substitutions at position 94
• Enhanced thermostability by introducing disulfide bridges between β-strands
• Improved catalytic efficiency at low pH via glutamate mutations in the active site

Rubisco Engineering Challenges

Diatom Rubisco exhibits a frustrating trade-off—while its specificity for CO₂ over O₂ is superior to terrestrial plants (SC/O ≈ 85 vs 80), its carboxylation rate remains suboptimal (k_cat^CO2 ≈ 3-5 s^-1). Current approaches include:

• Chimeric constructs combining diatom large subunits with proteobacterial small subunits
• Computational redesign of the L-subunit active site to reduce oxygenase activity
• Directed evolution under high-O₂/low-CO₂ selection pressure

The Silicon Connection: Frustule-Assisted Carbon Capture

Diatoms' unique silica cell walls (frustules) offer unexpected advantages for molecular carbon capture engineering:

Frustule Property	Carbon Capture Benefit
Nanoporous structure (2-50 nm pores)	Creatures molecular crowding effects that enhance enzyme-substrate encounters
Negative surface charge (-30 to -50 mV)	Concentrates positively charged bicarbonate ions near membrane transporters
Photonic crystal properties	Enhances light harvesting for photochemical reactions by 15-20%

Synthetic Biology Toolkits for Diatom Engineering

The development of genetic tools has transformed diatom manipulation from art to precision science:

• Golden Gate modular cloning: Enables rapid assembly of multigene constructs with >90% efficiency
• Crispr-Cas9 ribonucleoproteins: Achieves 30-80% editing efficiency in Phaeodactylum tricornutum
• TALE-activated promoters: Allows orthogonal control of carbon capture gene expression

The Single-Molecule Perspective: Tracking Enzymatic Performance

Cutting-edge single-molecule fluorescence techniques reveal surprising heterogeneity in diatom carbon capture proteins:

• FRET microscopy: Shows CA enzymes adopt multiple conformational states during catalysis
• Single-enzyme tracking: Reveals that 15% of Rubisco molecules account for 60% of total carboxylation activity
• Atomic force microscopy: Measures piconewton-scale forces during bicarbonate transporter operation

The Role of Protein Crowding and Phase Separation

The diatom pyrenoid—a liquid-like organelle where carbon fixation occurs—exhibits remarkable biophysical properties:

• Crowded protein concentrations (>300 mg/mL) enhance Rubisco activity by molecular confinement effects
• pH gradients across phase boundaries create microenvironments favoring HCO₃^- dehydration
• Intrinsically disordered linker proteins facilitate rapid enzyme reorganization in response to light changes

Mathematical Modeling of Enhanced Carbon Flux

The modified Michaelis-Menten equation accounting for diatom-specific conditions reveals optimization limits:

v = (Vmax[S]α)/(Km(1 + [O]/KO) + [S](1 + α))

Where:
α = 1 + ([H+]/KA1) + (KA2/[H+])
    

The model predicts that simultaneous optimization of CA and Rubisco could increase carbon fixation rates by 2.7-fold under saturating light conditions.

The Challenge of Metabolic Integration

Engineered proteins must function within the complex metabolic network of diatoms:

• The CCM consumes ~15% of cellular ATP under optimal conditions
• Silicification competes with carbon fixation for photosynthetic reductant
• Nitrogen limitation induces proteolytic degradation of non-essential enzymes

The Future of Molecular Carbon Capture Engineering

Emerging approaches promise to push diatom carbon capture beyond natural limits:

• Synthetic CO₂-fixing pathways: Implementing the CETCH cycle could bypass Rubisco limitations
• Quantum dot biohybrids: Nanocrystals conjugated to CA enhance light-driven proton pumping
• Spatial organization tools: Scaffolding enzymes into metabolons reduces substrate diffusion limits

The Grand Challenge: Scaling Molecular Solutions to Global Impact

The theoretical potential is staggering—a 50% improvement in diatom carbon fixation efficiency could sequester an additional 5 gigatons of CO₂/year. Yet formidable barriers remain:

• Ecological impacts: Engineered strains must not disrupt marine food webs
• Sinking rates: Carbon sequestration requires rapid transport to deep ocean layers
• Industrial cultivation: Photobioreactor designs must balance light penetration with high cell densities

The Path Forward: Integration Across Scales

The most promising solutions combine molecular, cellular, and ecosystem-level approaches:

1. Molecular: Continue protein engineering through computational design and directed evolution
2. Cellular: Develop synthetic organelles that spatially organize carbon capture enzymes
3. Cultivation: Engineer diatom consortia that share metabolic burdens across specialized strains
4. Ecosystem: Deploy controlled diatom blooms in high-nutrient, low-chlorophyll ocean regions