Engineering Synechocystis sp. PCC 6803 for Enhanced Bioplastic Production and CO₂ Reduction Under High pH Conditions
Yerin Cho, June Choi, Alyssa Han, Mirae Do, Yuri Kang, Minseo Kang, Seoyoon Chung, Kyungjin Oh, Jaeyun Kim, Charlotte Im, Jinah Chung, Joseph Park, Minjune Choi, Jay In Park, Ji Yoon Park, Jiyul Lee
iGEM KOREA-HS, Seoul, South Korea
Publication date: January 21, 2025
iGEM KOREA-HS, Seoul, South Korea
Publication date: January 21, 2025
DOI: http://doi.org/10.34614/JIYRC202437
ABSTRACT
The global challenges of climate change and plastic pollution necessitate innovative solutions that combine environmental and economic sustainability. This study focuses on engineering the unicellular cyanobacterium Synechocystis sp. PCC 6803 to enhance polyhydroxybutyrate (PHB) production while capturing atmospheric CO₂ under high pH conditions. Using adaptive laboratory evolution (ALE) to select for high pH tolerance, fluorescence-based quantification to assess PHB accumulation, and RNA sequencing (RNA-seq) analysis to investigate genetic adaptations, we developed Synechocystis strains capable of sustained growth and efficient PHB production under alkaline conditions. Our results identified pH 11.0 as the optimal environment for maximal PHB yield, with PHB content reaching 31.3 wt%, significantly higher than that observed at lower or more extreme pH levels. Fluorescence microscopy confirmed a strong correlation (R² = 0.9091) between intracellular PHB content and Nile red fluorescence intensity, validating this rapid quantification method for monitoring PHB levels. At pH 11.0, fluorescence images revealed a dense accumulation of PHB granules, indicating the efficacy of moderately alkaline conditions for PHB synthesis. RNA-seq analysis highlighted the metabolic shifts that underpin this adaptation, identifying 67 downregulated and 40 upregulated genes. Downregulated genes were associated with photosynthetic pathways and aminoacyl-tRNA biosynthesis, suggesting a rerouting of cellular resources away from growth and energy production. In contrast, the upregulated genes were predominantly linked to carbohydrate metabolism, specifically the fructose and mannose pathways, which likely contribute to increased precursor availability for PHB biosynthesis. This metabolic reprogramming enables Synechocystis sp. to efficiently convert CO₂ into bioplastics under stress, positioning it as a promising candidate for sustainable bioplastic production.
The global challenges of climate change and plastic pollution necessitate innovative solutions that combine environmental and economic sustainability. This study focuses on engineering the unicellular cyanobacterium Synechocystis sp. PCC 6803 to enhance polyhydroxybutyrate (PHB) production while capturing atmospheric CO₂ under high pH conditions. Using adaptive laboratory evolution (ALE) to select for high pH tolerance, fluorescence-based quantification to assess PHB accumulation, and RNA sequencing (RNA-seq) analysis to investigate genetic adaptations, we developed Synechocystis strains capable of sustained growth and efficient PHB production under alkaline conditions. Our results identified pH 11.0 as the optimal environment for maximal PHB yield, with PHB content reaching 31.3 wt%, significantly higher than that observed at lower or more extreme pH levels. Fluorescence microscopy confirmed a strong correlation (R² = 0.9091) between intracellular PHB content and Nile red fluorescence intensity, validating this rapid quantification method for monitoring PHB levels. At pH 11.0, fluorescence images revealed a dense accumulation of PHB granules, indicating the efficacy of moderately alkaline conditions for PHB synthesis. RNA-seq analysis highlighted the metabolic shifts that underpin this adaptation, identifying 67 downregulated and 40 upregulated genes. Downregulated genes were associated with photosynthetic pathways and aminoacyl-tRNA biosynthesis, suggesting a rerouting of cellular resources away from growth and energy production. In contrast, the upregulated genes were predominantly linked to carbohydrate metabolism, specifically the fructose and mannose pathways, which likely contribute to increased precursor availability for PHB biosynthesis. This metabolic reprogramming enables Synechocystis sp. to efficiently convert CO₂ into bioplastics under stress, positioning it as a promising candidate for sustainable bioplastic production.
