Redirecting photosynthetic electron flux in the cyanobacterium Synechocystis sp. PCC 6803 by the deletion of flavodiiron protein Flv3

Abstract

BackgroundOxygen-evolving photoautotrophic organisms, like cyanobacteria, protect their photosynthetic machinery by a number of regulatory mechanisms, including alternative electron transfer pathways. Despite the importance in modulating the electron flux distribution between the photosystems, alternative electron transfer routes may compete with the solar-driven production of CO2-derived target chemicals in biotechnological systems under development. This work focused on engineered cyanobacterial Synechocystis sp. PCC 6803 strains, to explore possibilities to rescue excited electrons that would normally be lost to molecular oxygen by an alternative acceptor flavodiiron protein Flv1/3—an enzyme that is natively associated with transfer of electrons from PSI to O2, as part of an acclimation strategy towards varying environmental conditions.ResultsThe effects of Flv1/3 inactivation by flv3 deletion were studied in respect to three alternative end-products, sucrose, polyhydroxybutyrate and glycogen, while the photosynthetic gas fluxes were monitored by Membrane Inlet Mass Spectrometry (MIMS) to acquire information on cellular carbon uptake, and the production and consumption of O2. The results demonstrated that a significant proportion of the excited electrons derived from photosynthetic water cleavage was lost to molecular oxygen via Flv1/3 in cells grown under high CO2, especially under high light intensities. In flv3 deletion strains these electrons could be re-routed to increase the relative metabolic flux towards the monitored target products, but the carbon distribution and the overall efficiency were determined by the light conditions and the genetic composition of the respective pathways. At the same time, the total photosynthetic capacity of the Δflv3 strains was systematically reduced, and accompanied by upregulation of oxidative glycolytic metabolism in respect to controls with the native Flv1/3 background.ConclusionsThe observed metabolic changes and respective production profiles were proposedly linked with the lack of Flv1/3-mediated electron transfer, and the associated decrease in the intracellular ATP/NADPH ratio, which is bound to affect the metabolic carbon partitioning in the flv3-deficient cells. While the deletion of flv3 could offer a strategy for enhancing the photosynthetic production of desired chemicals in cyanobacteria under specified conditions, the engineered target pathways have to be carefully selected to align with the intracellular redox balance of the cells.

Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes

“…Computational 40,41 and experimental 42,43 studies have shown that efficient production of some biochemicals would require lowering the ATP/NADPH ratio in the cyanobacteria cell. The was shown to lower cellular ATP/NADPH ratio in Synechocystis 43 . Finally, the CCA-tRNA nucleotidyltransferase pcnB, essential for tRNA maturation and thus protein synthesis, was also enriched.…”

On the Role and Production of Polyhydroxybutyrate (PHB) in the Cyanobacterium Synechocystis sp. PCC 6803

“…One example for such a mechanism is the flavodiiron protein Flv3, which serves as a sink for excess electrons from the photosynthetic light reaction, by converting O 2 to H 2 O. A recent publication showed indeed increased PHB synthesis in a Flv3 deficient strain [33]. If PHB is indeed serving as an intracellular electron sink, its absence might be compensated by a higher activity of Flv3.…”

“…It can be assumed that the correct regulation of the ATP/NADPH ratio is crucial for cyanobacterial cells. Since the production of PHB from glycogen provides ATP but consumes NADPH, the biopolymer could help the cells to regulate this ratio under conditions of electron excess [33].…”

From gas to sugar: Trehalose production inCupriavidus necatorfrom CO2and hydrogen gas

“…Recently, autotrophic sugar production and secretion have been engineered and demonstrated in cyanobacteria by various studies (Ducat et al, 2012;Lin et al, 2020;Thiel et al, 2019). An impressive proportion of the fixed carbon can be directed to sugar synthesis and exported from the cell.…”