Engineering a Central Carbon Metabolism Pathway to Increase the Intracellular Acetyl-CoA Pool in Synechocystis sp. PCC 6803 Grown under Photomixotrophic Conditions.
2021
In cyanobacteria, photomixotrophic growth is considered as a promising strategy to achieve both high cell density and product accumulation. However, the conversion of glucose to acetyl coenzyme A (acetyl-CoA) in the native glycolytic pathway is insufficient, which decreases the carbon utilization and productivity of engineered cyanobacteria under photomixotrophic conditions. To increase the carbon flux from glucose to key intracellular precursor acetyl-CoA in Synechocystis sp. PCC 6803 (hereafter, Synechocystis 6803) under photomixotrophic conditions, a synthetic nonoxidative cyclic glycolysis (NOG) pathway was introduced into the wild type strain, which successfully increased the intracellular pool of acetyl-CoA by approximately 1-fold. To minimize the competition for glucose, the native Embden-Meyerhof-Parnas (EMP) and Entner-Doudoroff (ED) pathways were knocked out, respectively. Notably, eliminating the native ED pathway in the engineered strain carrying the NOG pathway further increased the intracellular pool of acetyl-CoA up to 2.8-fold. Another carbon consuming pathway in Synechocystis 6803, the glycogen biosynthesis pathway, was additionally knocked out in the above-mentioned engineered strain, which enabled an increase of the intracellular acetyl-CoA pool by up to 3.5-fold when compared with the wild type strain. Finally, the content of intracellular lipids was analyzed as an index of the productive capacity of the engineered Synechocystis 6803 cell factory under photomixotrophic conditions. The results showed the total lipids yield increased about 26% compared to the wild type (from 15.71% to 34.12%, g/g glucose), demonstrating that this integrated approach could represent a general strategy not only for the improvement of the intracellular concentration of acetyl-CoA, but also for the production of value-added chemicals that require acetyl-CoA as a key precursor in cyanobacteria.
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