Engineering of Synthetic Reverse Glyoxylate Shunt Module for enhanced carbon incorporation in cyanobacteria
- Author(s): Li, Xiaoqian
- Advisor(s): Liao, James C
- et al.
Synthetic Biology and Metabolic engineering research aim to elucidate the underlying principle of biological systems and enable a predicable cellular behavior through artificial design. In this work, the discipline of synthetic biology was applied to photosynthetic organism to improve carbon intake efficiency.
After survey and analysis of existing carbon fixation metabolic pathways, the design of a synthetic Reverse Glyoxylate Shunt (rGS) core module was invented. This core module could potentially pair with multiple metabolic routes to generate various synthetic carbon incorporation/fixation pathways. The design of rGS-Glycerate pathway, which could theoretically double the Acetyl-CoA yields from C3 metabolites, was reduced to practice at first due to its technical feasibility simulated through computation. The performance of rGS-Glycerate pathway reached its theoretical value in vitro. The pathway was further proved to be functional in E.coli through growth auxotroph test. Finally, rGS-Glycerate pathway was implemented into a model photosynthetic microorganism, Synechococcus elongatus PCC7942, after iterative “Design-Build-Test” engineering cycles. The higher Acetyl-CoA yield was characterized in terms of higher production of ketoisocaproate (KIC) that required Acetyl-CoA as its precursor in cyanobacteria. Further metabolomics analysis found out that one of the most abundant fatty acids, Oleic acids, reached to 2.5 folds of its content in WT. Another rGS core module contained pathway, rGS-Serine pathway, was also constructed in Synechococcus elongatus PCC7942. Similar level of KIC production was detected in those rGS-Serine strains as well.
In sum, this work is the initial demonstration that the rGS core module contained pathways were compatible with photosynthetic machinery and could potentially lead to enhanced carbon incorporation in photosynthetic organisms.