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

Abstract

Background Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear. Results Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains. Conclusions The developed approach, based on parallel 13C tracer studies with GC–MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments.

Entner-Doudoroff pathway in Synechocystis PCC 6803: Proposed regulatory roles and enzyme multifunctionalities

“…The possible explanations are: i) a minor flux via ED-P, as suggested in our own flux estimation, ii) significant flux under certain conditions, such as fluctuating light ( Schulze et al, 2022 ), which may explain previously reported significant level of KDPG ( Chen et al, 2016 ), otherwise undetectable ( Will et al, 2019 ; Schulze et al, 2022 ); we note that 10% fluctuations in light (i.e., ATP and NADPH regeneration in the model) did not influence our flux estimations (data not shown) for Δ eda , or iii) ED-P is missing in Synechocystis . Furthermore, Δ eda was reported to deactivate OPP shunt ( Schulze et al, 2022 ), which disagrees with our model prediction purely based on the metabolic flux via ED-P. Thus, Δ eda mediated deactivation of OPP could support either of the presented hypotheses, i.e., role of KDPG in carbon catabolite repression or the other role of EDA in the proximity of TCA cycle might play a role in regulating OPP as neither of these scenarios are in the model.…”

“…This study ( Schulze et al, 2022 ) compared WT and Δ eda , however, the authors could not detect any flux via ED-P. The possible explanations are: i) a minor flux via ED-P, as suggested in our own flux estimation, ii) significant flux under certain conditions, such as fluctuating light ( Schulze et al, 2022 ), which may explain previously reported significant level of KDPG ( Chen et al, 2016 ), otherwise undetectable ( Will et al, 2019 ; Schulze et al, 2022 ); we note that 10% fluctuations in light (i.e., ATP and NADPH regeneration in the model) did not influence our flux estimations (data not shown) for Δ eda , or iii) ED-P is missing in Synechocystis . Furthermore, Δ eda was reported to deactivate OPP shunt ( Schulze et al, 2022 ), which disagrees with our model prediction purely based on the metabolic flux via ED-P.…”

“…In the case of Synechocystis, there is no mention of gluconate as a substrate for ilvD in recent biochemical analysis (Zhang et al, 2020). Nevertheless, a product of EDD, KDPG, has been detected in a single study (Chen et al, 2016) while determined to be undetectable in others (Will et al, 2019;Schulze et al, 2022). The situation is further complicated by an approximately 30-fold difference in 6-phosphogluconate (a substrate of EDD) level under autotrophic vs. mixotrophic conditions (Yoshikawa et al, 2013), which might be the reason for the elusiveness of KDPG in metabolic profiling.…”

“…However, for Δ eda , the model matches the reported flux via OPP-P (as well as via PKET-P) (WT) ( Figure 2 vs. Table 4 ). Since ED-P and OPP-P are closely connected ( Figure 1 ) and ED-P was either not considered in the previous fluxomic analyses ( Nakajima et al, 2014 ), or the flux via ED-P was not detected and assumed too low ( Schulze et al, 2022 ). Thus, our simulation offers both an alternative solution and a possible revision of the available fluxomic data.…”

“…We did not perform any simulations based on the signaling role of KDPG as the growth impairment caused by Δ eda provides a sufficient description only of its impact, but new experimental data are needed to determine the kinetics of the signaling process. The current version of the model assumes a very low level of KDPG for WT, based on reported undetectable levels ( Will et al, 2019 ; Schulze et al, 2022 ).…”