[P004] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P02-188] Enhancing CO₂ Conversion Efficiency for Biological Carbon-Negative Technologies

○Jo-Yu Wu¹, Emma J. Huang¹, Chih-Ting Chang¹, Paul P. Lin¹ (1. National Yang Ming Chiao Tung University (Taiwan))

Keywords:

Cupriavidus necator,non-oxidative glycolysis pathway,carbon conversion efficiency,Cre-lox gene editing,biological carbon fixation

Anthropogenic CO₂ emissions have reached unprecedented levels, driving global warming and extreme weather. Carbon capture and utilization (CCU) is a key strategy for reducing atmospheric CO₂ and converting it into valuable products. Among various CCU approaches, biological carbon fixation offers unique advantages. However, current biological carbon-negative technologies are limited by insufficient conversion efficiency from CO₂ to target products.Biological carbon fixation via the Calvin–Benson–Bassham (CBB) cycle is widespread in nature. In this cycle, three molecules of CO₂ are fixed by RuBisCO to produce one molecule of glyceraldehyde 3-phosphate (G3P). G3P then enters the Embden–Meyerhof–Parnas (EMP) pathway to generate three-carbon (C3; e.g., pyruvate) and two-carbon (C2; e.g., acetyl-CoA) metabolites, which serve as key precursors for biosynthetic and energy metabolism. However, the CBB cycle converts six molecules of CO₂ into two molecules of acetyl-CoA while releasing two molecules of CO₂, limiting the theoretical CO₂-to-acetyl-CoA conversion efficiency to 67%. To overcome this limitation, we aim to integrate the CBB cycle with the non-oxidative glycolysis (NOG) pathway¹ in Cupriavidus necator (C. necator), thereby bypassing carbon loss and increasing the theoretical carbon conversion efficiency toward 100%.To achieve this goal, C. necator was metabolically engineered to integrate the CBB cycle with NOG pathways to convert acetyl-CoA into ethanol. We applied a rapid screening strategy to avoid potential kinetic traps caused by F/Xpk activity. To this end, 23 plasmids featuring various promoters were designed and cloned; 10 of these were successfully transformed into C. necator. The top-performing strain, EH16-6, was identified via small-scale fermentation. To further optimize production, we developed a Cre-lox-based genetic editing system for markerless gene deletion. We subsequently deleted phaC using this method to eliminate PHB production and redirect metabolic resources.