Engineering a Central Carbon Metabolism Pathway to Increase the Intracellular Acetyl-CoA Pool in Synechocystis sp. PCC 6803 Grown under Photomixotrophic Conditions

Abstract

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.

GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways

“…Photomixotrophic metabolism is particularly promising for the future commercialization of cyanobacteria because the co-utilization of CO 2 and organic carbon provides additional building blocks and energy. This mode enables enhanced production and higher cell density [ 61 ] and confers industrial feasibility [ 62 ]. Remarkably, photomixotrophic production has increased titers up to five-fold over traditional autotrophic conditions [ 63 ].…”

Transcriptomics and metabolomics of engineered Synechococcus elongatus during photomixotrophic growth

“…Glycolysis is a primary pathway for glucose metabolism in microorganisms and could be affected in YQ2-gal while utilizing glucose [ 19 , 20 ]. Genes in glycolysis were not differentially expressed in YQ2-gal compared with WT, while increased abundances of phosphorylated metabolites in glycolysis, such as glucose 6-phosphate (G-6-P), fructose 6-phosphate (F-6-P) and fructose 1,6-bisphosphate (F-1, 6-BP), were detected (Fig.…”

Cross-Feeding between Cyanobacterium Synechococcus and Escherichia Coli in Artificial Autotrophic-Heterotrophic Co-Culture System Revealed by Integrated Omics Analysis

“…Liquid chromatography-mass spectrometry (LC-MS) based targeted metabolomics was performed according to the protocol described previously 29 . Cells (5 OD 730 unit) were harvested at 48 h via centrifugation at 7380 rpm for 5 min at 4 °C (Eppendorf 5430R), quenched, and extracted rapidly with 900 μL of 80:20 methanol/water (v/v; -80 °C pretreated) and then frozen in liquid nitrogen.…”

“…As the stability and productivity in the co-culture system was dependent on the cyanobacterial sucrose production, intracellular levels of key metabolites within Synechococcus cscB + cells were investigated. LC-MS based metabolomics approach, which has been employed previously to comparatively analyze cellular metabolism in the engineered cyanobacterial strains 29,36 , was applied to compare co-cultivated and pure cultural Synechococcus cscB + . As shown in Fig.…”