Triacylglycerol accumulation in Rhodococcus opacus DSM 43205 from Knallgas and Knallgas-derived soluble intermediates

Rhodococcus opacus DSM 43205 combines broad substrate versatility with autotrophic growth on Knallgas (H₂/O₂/CO₂), making it a promising chassis for carbon-efficient triacylglycerol (TAG) production. We quantified growth, intracellular TAG content, yields, specific productivities, and fatty-acid profiles during nitrogen-limited accumulation on Knallgas and Knallgas-derived intermediates (methanol, acetate, 1-propanol, butyrate, valerate); glucose served as a reference. Acetate yielded the highest TAG content (0.580 ± 0.095 g gCDW⁻¹) and approached its theoretical maximum (96 %), whereas methanol (0.305 ± 0.014) and Knallgas (0.325 ± 0.047) were lowest. Propanol and valerate drove > 80 % odd-chain fatty acids via propionyl-CoA priming. A reduced central-metabolism flux balance analysis (FBA) model reproduced substrate-specific NADPH strategies and guided engineering hypotheses: a RuMP cycle for methanol raised the theoretical TAG yield to 0.327 g g⁻¹ ; bypassing pyruvate decarboxylation and expression of a NAD(P)⁺ dependent transhydrogenase under Knallgas further improved hydrogen yields to 1.083 g g⁻¹. Extending the system boundary to upstream chemical synthesis showed that producing 1 kg TAG required 0.95 kg H₂ and 8.64 kg CO₂ via acetate, versus 2.56 kg H₂ and 5.95 kg CO₂ for direct Knallgas fermentation. The most efficient H₂ utilization was predicted for methanol using the RuMP cycle (0.62 kg H₂ per kg TAG). Increases in TAG-free biomass and direct assays revealed substantial cell-bound carbohydrate and soluble EPS formation on glucose and Knallgas. Quantitatively explaining differences between experimental and theoretical yields. The curated FBA model and Escher-ready map are released openly, enabling community-driven metabolic engineering and process design for carbon-negative lipids.