Double blocking of carbon metabolism causes a large increase of Calvin–Benson cycle compounds in cyanobacteria

Double blocking of carbon metabolism causes a large increase of Calvin–Benson cycle compounds in cyanobacteria María Teresa Domínguez-Lobo , 1 Miguel Roldán , 1,2 Alba María Gutiérrez-Diánez , 1,2 Francisco Javier Florencio 1,2 and María Isabel Muro-Pastor 1, * 1 Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), CSIC-Universidad de Sevilla, Sevilla 41092, Spain 2 Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Biología, Universidad de Sevilla, Sevilla 41012, Spain *Author for correspondence: [email protected] The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) is: María Isabel Muro-Pastor. Abstract Carbon-flow-regulator A (CfrA) adapts carbon flux to nitrogen conditions in nondiazotrophic cyanobacteria. Under nitrogen deficiency, CfrA leads to the storage of excess carbon, which cannot combine with nitrogen, mainly as glycogen. cfrA overexpression from the arsenite-inducible, nitrogen-independent P arsB promoter allows analysis of the metabolic effects of CfrA accumulation. Considering that the main consequence of cfrA overexpression is glycogen accumulation, we examined carbon distribution in response to cfrA expression in Synechocystis sp. PCC 6803 strains impaired in synthesizing this polymer. We carried out a comparative phenotypic analysis to evaluate cfrA overexpression in the wild-type strain and in a mutant of ADP-glucose pyrophosphorylase (ΔglgC), which is unable to synthesize glycogen. The accumulation of CfrA in the wildtype background caused a photosynthetic readjustment although growth was not affected. However, in a ΔglgC strain, growth decreased depending on CfrA accumulation and photosynthesis was severely affected. An elemental analysis of the H, C, and N content of cells revealed that cfrA expression in the wild-type caused an increase in the C/N ratio, due to decreased nitrogen assimilation. Metabolomic study indicated that these cells store sucrose and glycosylglycerol, in addition to the previously described glycogen accumulation. However, cells deficient in glycogen synthesis accumulated large amounts of Calvin–Benson cycle intermediates as cfrA was expressed. These cells also showed increased levels of some amino acids, mainly alanine, serine, valine, isoleucine, and leucine. The findings suggest that by controlling cfrA expression, in different conditions and strains, we could change the distribution of fixed carbon, with potential biotechnological benefits. Open Access Received January 08, 2024. Accepted January 19, 2024. Advance access publication February 20, 2024 © The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Research Article Introduction Cyanobacteria, as photosynthetic organisms, have great potential for the sustainable production of different compounds of biotechnological interest (Tan et al. 2022; Toepel et al. 2023). The fundamental advantage of these organisms lies in their ability to transform CO 2 into high-value products using solar energy. They present a high adaptive capacity based on their great metabolic plasticity (Xiong et al. 2017). An exhaustive characterization of cyanobacterial physiology and metabolism is key to the development of any biotechnological strategy. The model cyanobacterium Synechocystis sp. PCC 6803 (hereinafter Synechocystis) has been extensively studied and its global metabolism has been recently reviewed (Mills et al. 2020). In relation to carbon metabolism, recent studies have analyzed and https://doi.org/10.1093/plphys/kiae083 PLANT PHYSIOLOGY 2024: 195: 1491–1505 Downloaded from https://academic.oup.com/plphys/article/195/2/1491/7611675 by Biblioteca de la Universidad de Sevilla user on 05 June 2024 quantified the contribution of different metabolic pathways under different conditions (Doello et al. 2018; Shinde et al. 2020; Lucius et al. 2021; Schulze et al. 2022; Lu et al. 2023). Glycogen is the main carbon storage compound in cyanobacteria. The metabolism of this polymer contributes to growth in diurnal cycles and survival under different stress conditions (Cano et al. 2018; Luan et al. 2019; Ortega-Martínez et al. 2022). Recently, glycogen metabolism has been identified as an essential element for optimal cyanobacterial growth in the rapid light–dark cycle of low-Earth orbit, which is relevant for space travel (Bishé et al. 2023). Numerous works have tried to alter the metabolism of glycogen in cyanobacteria, either by increasing or decreasing its synthesis, in order to redirect the fixed carbon toward certain pathways of interest (Davies et al. 2014; Li et al. 2014; Sun et al. 2018; Velmurugan and Incharoensakdi 2018; Mittermair et al. 2021; Sivaramakrishnan and Incharoensakdi 2022). These engineering strategies of glycogen metabolism and their physiological effects in cyanobacteria have been reviewed (Luan et al. 2019). On many occasions, the target selected for these manipulations is the glucose-1-phosphate adenylyltransferase (glgC) gene that encodes ADP-glucose pyrophosphorylase (AGP). This enzyme generates ADP-glucose from glucose-1-phosphate and ATP and is generally considered the rate-limiting step that holds control over the entire glycogen accumulation process (Gründel et al. 2012). Knockout mutants of glgC are completely unable to synthesize glycogen as well as other compounds that depend on ADP-glucose for their synthesis, such as the osmolyte glucosylglycerol (GG) (Klähn and Hagemann 2011). In Synechocystis, the glgC mutants grow photoautotrophically in continuous light in a very similar way to the wild-type (WT) strain. However, they show a pleiotropic phenotype in other culture conditions, with loss of viability in light–dark cycles and an anomalous response to nitrogen deficiency (Carrieri et al. 2012, 2017; Gründel et al. 2012). This response includes a metabolic carbon overflow, in which part of the fixed carbon is directed toward the synthesis and excretion of organic acids, mainly pyruvate and 2-oxoglutarate (2-OG). This excretion is affected by light intensity and can also be triggered when nitrogen is available. Under an excess energy input, it is considered an alternative energy dissipation mechanism when the glycogen buffer is not present (Cano et al. 2018). Carbon-flow-regulator A (CfrA) is a regulatory protein, highly conserved in cyanobacteria, involved in the adaptation of carbon flow to nitrogen deficiency. Its expression increases very markedly in Synechocystis during nitrogen control A (NtcA)-dependent acclimation to nitrogen starvation (Giner-Lamia et al. 2017; Muro-Pastor et al. 2020). During this adaptive response (chlorosis), a large amount of carbon is stored as glycogen. CfrA, also called PII-interacting regulator of carbon metabolism (PirC), facilitates this accumulation by inhibiting 3-phosphoglycerate mutase (PGAM), an enzyme whose activity directs fixed carbon toward lower glycolysis. CfrA also interacts with the PII regulatory protein, mainly as a function of the binding of 2-OG to the latter, and thus affects the CfrA/PGAM interaction (Orthwein et al. 2021). The overexpression of cfrA triggers the accumulation of glycogen even in the presence of a nitrogen source and regardless of the presence of PII. The cells reach a pseudo-chlorotic state with low levels of pigments but without growth arrest. In the case of nitrogen depletion, cfrA overexpressing strains respond in a similar way to the WT strain, developing chlorosis; however, the readdition of nitrogen does not allow the process to be reversed and the concomitant consumption of accumulated glycogen that takes place in the WT strain. On the contrary, the strains lacking CfrA revert chlorosis in an accelerated manner compared to the WT strain. They present a greater flow of carbon toward the lower glycolysis and the TCA cycle for its combination with nitrogen and the synthesis of proteins (Muro-Pastor et al. 2020). Consistent with these results, CfrA-deficient strains have been metabolically engineered to maximize polyhydroxybutyrate production under nitrogendeficient conditions. This polymer is synthesized naturally in Synechocystis from acetyl-CoA (Ac-CoA), and therefore, its synthesis is favored in the absence of CfrA (Koch et al. 2020; Orthwein et al. 2021). Given these results, the modulation of cfrA expression could have an important influence on the distribution of endogenous carbon. Taking into account that CfrA promotes the storage of carbon as glycogen, we wonder what would be the effect of the overexpression of cfrA in a mutant strain ΔglgC, unable to synthesize this polymer. As mentioned above, the glgC mutation causes a metabolic overflow with accumulation of pyruvate and 2-oxoglutarate. The inhibition of PGAM mediated by CfrA in combination with the glgC mutation could give rise to an unexplored carbon redistribution. We have undertaken a comparative analysis of the overexpression of cfrA in a WT or a ΔglgC genetic background. In a WT strain, the overexpression of cfrA leads to a massive accumulation of glycogen and other carbon compounds such as sucrose or GG, without removal of the nitrogen source from the medium. However, in a ΔglgC strain, the accumulation of CfrA has drastic negative effects on photosynthetic activity and growth. The carbon is reallocated to different carbohydrates and specific amino acids like alanine, serine, or leucine. Results cfrA overexpression severely affects the growth of ΔglgC mutants To analyze the phenotype associated with cfrA overexpression in strains unable to synthesize glycogen, the previously described P arsB -controlled version of cfrA was introduced into a ΔglgC strain. In the resulting strain (ΔglgC/Pars-cfrA), the endogenous cfrA locus was eliminated as previously reported (Muro-Pastor et al. 2020). To rule out a deleterious effect of arsenite itself, the growth of the parental strains WT and ΔglgC was compared with those that overexpress cfrA. The addition of arsenite (500 µM) only substantially affects the growth of the ΔglgC/Pars-cfrA strain (Supplementary Fig. S1). To further characterize the arsenite-dependent accumulation of CfrA 1492 |PLANT PHYSIOLOGY 2024: 195; 1491–1505 Domínguez-Lobo et al. Downloaded from https://academic.oup.com/plphys/article/195/2/1491/7611675 by Biblioteca de la Universidad de Sevilla user on 05 June 2024 and its effect on the ΔglgC/Pars-cfrA strain, it was first propagated in flasks using BG11C medium without arsenite, and then 150-ml cultures were inoculated under standard conditions in BG11C at 0.4 OD 750 and supplemented with 1% (v/v) CO 2 . When these cultures reached ∼1 OD 750 , increasing amounts of arsenite were added to each culture (20, 50, 200, and 500 µM). A control culture without arsenite was also included. As shown in Fig. 1A, the growth of the ΔglgC/ Pars-cfrA strain was severely affected depending on the amount of arsenite added, with almost no growth at concentrations above 50 µM. Considering the characteristics of the P arsB promoter, the transcription of cfrA gene should stop in the A D E C B Figure 1. Analysis of the cfrA overexpressing strains Pars-cfrA and ΔglgC/Pars-cfrA. A) Growth analysis of ΔglgC/Pars-cfrA strain before and after the addition of the indicated amounts of arsenite. B) Growth of strain ΔglgC/Pars-cfrA after eliminating the arsenite present in the precultures at the indicated concentrations. C) Western-blot analysis of CfrA in ΔglgC/Pars-cfrA strain during the process of addition and subsequent removal of arsenite. Total protein crude extracts were obtained from cells corresponding to 2 OD 750 , resolved on SDS–PAGE, blotted and incubated with anti-CfrA antibodies. As a control for protein loading, membranes were incubated also with anti-GroEL antibodies. D) Growth analysis of Pars-cfrA strain before and after the addition of the indicated amounts of arsenite. E) Glycogen content of Pars-cfrA strain at 0, 24, and 48 h after arsenite addition at the indicated concentrations. Error bars in A), B), D), and E) represent SD of the mean values from 3 independent experiments. Carbon metabolism in cfrA overexpressors PLANT PHYSIOLOGY 2024: 195; 1491–1505 |1493 Downloaded from https://academic.oup.com/plphys/article/195/2/1491/7611675 by Biblioteca de la Universidad de Sevilla user on 05 June 2024 ΔglgC/Pars-cfrA strain by inducer removal. To analyze whether the cells were able to resume growth when the arsenite was removed, it was eliminated after 48 h of culture. The cells were washed and recultured without arsenite, setting the OD 750 of all cultures to 1 at the beginning of the experiment. The OD 750 and the CfrA amount in the cells were monitored. As shown in Fig. 1B, the cells recover growth depending on the amount of arsenite in the previous cultures, which determines the level of CfrA accumulation of the cells. Thus, cultures with lower concentrations of arsenite resumed growth earlier. CfrA accumulated soon after arsenite addition in the ΔglgC/ Pars-cfrA strain, and it progressively disappeared from the cells when eliminating the arsenite in a way that depended on the concentration used in each culture (Fig. 1C). Cells from the control culture without arsenite showed slower growth in the reculture than would be expected since they did not accumulate CfrA. This behavior is most likely due to the fact that these cells have maintained active growth in the previous culture and are in the stationary phase at the beginning of the recultivation. As previously described, the expression of cfrA by the addition of arsenite did not substantially affect the growth of the Pars-cfrA strain, observing only a slight increase in the OD 750 as a function of the added arsenite (Fig. 1D) and a concentration-dependent accumulation of glycogen (Fig. 1E). cfrA overexpression in ΔglgC genetic background negatively affects the photosynthetic activity The drastic effect that CfrA produces on the growth of the ΔglgC/Pars-cfrA strains led us to evaluate its photosynthetic capacity, from oxygen evolution rates, at different times after arsenite addition. As a control, the ΔglgC strain was analyzed under the same conditions. Oxygen-evolving rates at different light intensities were determined without inducer (0 h) and at 8 or 24 h after arsenite addition (50 µM) (Fig. 2, A to C). After taking the initial samples, the culture of each strain was divided into 2, one was kept without the addition of inducer and the other was added arsenite. While before the addition of arsenite, the saturation curves were similar in the 2 strains analyzed (Fig. 2A), after the addition of the inducer, the light saturation curve of ΔglgC/Pars-cfrA strain reached a 3-fold lower maximum O 2 evolution rate (Fig. 2, B and C). We also measured PSII operating efficiency [Y(II)] by pulse-amplitude- modulation fluorometry. Accordingly with the measurements of oxygen evolution, PSII efficiency strongly decreased in A B C D Figure 2. Effects of cfrA overexpression on photosynthetic parameters of ΔglgC/Pars-cfrA strain. A to C) Light saturation curves of cells from ΔglgC/ Pars-cfrA strain and the control strain ΔglgC, normalized to chlorophyll content, measured before (0 h) or after (8 and 24 h) arsenite addition (50 µM). D) PSII operating efficiency [Y(II)] of ΔglgC/Pars-cfrA and ΔglgC strains determined under growth light (60 µmol photons m −2 s −1 ) before (0 h) or after (8 and 24 h) arsenite addition (50 µMM). Data are means ± SD from 3 biological replicates in all cases. 1494 |PLANT PHYSIOLOGY 2024: 195; 1491–1505 Domínguez-Lobo et al. Downloaded from https://academic.oup.com/plphys/article/195/2/1491/7611675 by Biblioteca de la Universidad de Sevilla user on 05 June 2024 ΔglgC/Pars-cfrA strain after arsenite addition (Fig. 2D). Given that in a WT genetic background for glycogen synthesis (Pars-cfrA strain), the expression of cfrA does not affect growth, we decided to analyze the photosynthetic activity of this strain after the addition of arsenite. In previous works, we had observed that the addition of 1 mM of arsenite to this strain caused a significant decrease in photosynthetic pigments, chlorophyll, and phycocyanin (Muro-Pastor et al. 2020). Taking these data into account, the O 2 evolution rate of this strain was analyzed 24 h after arsenite addition (50 µM or 1 mM), using the WT strain as control. After the addition of arsenite, a decrease in the evolution of oxygen was observed in the Pars-cfrA strain, especially in the cultures treated with 1 mM of inducer (Fig. 3A). These treatments did not affect the oxygen evolution of the WT strain (Fig. 3B). However, when the oxygen evolution measurements were related to the amount of chlorophyll (Supplementary Fig. S2), both strains presented a similar rate, independent of the arsenite addition (Fig. 3, C and D), suggesting that the observed effect in the Pars-cfrA strain was due to a readjustment in the content of photosynthetic pigments. CfrA interacts with PGAM under nitrogen-replete conditions The CfrA/PGAM interaction was demonstrated under nitrogen-deficient conditions, when cfrA is naturally expressed (Orthwein et al. 2021). We wanted to check here if this interaction also took place in our working conditions when cfrA was overexpressed in different genetic backgrounds under nitrogen-replete conditions. To this end, coimmunoprecipitation experiments were conducted. As PII protein is considered, the major CfrA interaction partner (Orthwein et al. 2021), a previously described PII lacking strain ΔglnB/Pars-cfrA was included, in order to promote the CfrA/PGAM interaction. All the phenotypic traits associated with the cfrA overexpression in nitrogen-replete conditions are maintained and even intensified in this strain (Muro-Pastor et al. 2020). Cells from the different strains (Pars-cfrA, ΔglgC/Pars-cfrA, and ΔglnB/Pars-cfrA), cultivated in the presence of arsenite for 24 h, were used to obtain crude extracts. CfrA was immunoprecipitated using anti-CfrA antibodies coupled to protein A superparamagnetic beads. Controls with preimmune serum were carried out in parallel. Precipitated material was analyzed by LC-MS/MS using a TOF quadrupole and ProteinPilot v5.0.1 software for protein identification. A list of the first 30 proteins identified only with the immune serum and ordered by their score is available for each cfrA overexpressing strain. The score is based on the number of distinctive peptides identified for each protein and the false discovery rate (Supplementary Data Set S1). 2,3-Phosphoglycerate-independent PGAM coimmunoprecipitated with CfrA, only with the immune serum, in all the strains analyzed. This interaction was more robust in A B C D Figure 3. Effects of cfrA overexpression on photosynthetic parameters of Pars-cfrA strain. A) Light saturation curves of cells from Pars-cfrA strain equalized to OD 750 of 1 at 24 h after arsenite addition (50 µM or 1 mM). A control without arsenite was included. B) Light saturation curves of cells from WT strain in the same conditions. C) Light saturation curves of cells from Pars-cfrA strain 24 h after arsenite addition (50 µM or 1 mM) normalized to chlorophyll content. D) Light saturation curves of cells from WT strain in the same conditions, normalized to chlorophyll content. Data are means ± SD from 3 biological replicates in all cases. Carbon metabolism in cfrA overexpressors PLANT PHYSIOLOGY 2024: 195; 1491–1505 |1495 Downloaded from https://academic.oup.com/plphys/article/195/2/1491/7611675 by Biblioteca de la Universidad de Sevilla user on 05 June 2024 terms of score and number of peptides identified in the case of the strain lacking PII. These results clearly indicate that CfrA is acting through its interaction with PGAM in our working conditions. CfrA accumulation impact on intracellular element stoichiometry Taking into account the changes in the content of photosynthetic pigments and total protein associated with cfrA overexpression (Muro-Pastor et al. 2020), we set out to analyze the elemental composition of the biomass from Pars-cfrA and ΔglgC/Pars-cfrA strains as a function of CfrA accumulation. To eliminate potential interferences due to the addition of arsenite, the WT and ΔglgC strains were analyzed as controls. All the strains were grown under standard conditions, and cfrA expression was induced as in previously described experiments using 100 µM arsenite. As shown in Fig. 4A, a slight and progressive increase in the C/N ratio (on a % dry mass basis) was observed in the WT strain after arsenite addition. This increase (13% at the end of the time analyzed) was most likely due to the age of the cultures, which leads to some storage of glycogen. A much more pronounced increase in the C/N ratio (24%) was observed for the strain Pars-cfrA already at 8 h after the addition of arsenite, reaching an increase of 53% 48 h after induction. However, in strains unable to synthesize glycogen, no substantial change in the C/N ratio was observed after the addition of arsenite. The observed effect on the C/N ratio is due in all cases to a decrease in nitrogen content rather than a change in total carbon (Fig. 4, B and C). These data agree with a clear decrease in nitrogen assimilation in the Pars-cfrA strain, as can be seen by the decrease in metabolites related to the GS-GOGAT cycle and the ornithine–ammonium cycle (OAC) such as glutamine, arginine, citrulline, aspartate, and proline. The glutamate did not present changes (Fig. 5). Metabolic analysis reveals clear differences in carbon partitioning associated with cfrA overexpression as a function of glycogen metabolism In order to analyze the metabolic effects of cfrA overexpression in the different genetic backgrounds, we carried out a comparative global metabolomic study between the Pars-cfrA and ΔglgC/Pars-cfrA strains in a time course after the addition of arsenite (50 µM). WT and ΔglgC strains A B C Figure 4. Chemical elemental analysis of the cfrA overexpressing strains Pars-cfrA and ΔglgC/Pars-cfrA, together with the control strains WT and ΔglgC. A) Elemental intracellular ratio of C/N, on a % dry mass basis, of the different strains analyzed before and after arsenite addition (100 µM). B) Nitrogen content of the different strains analyzed before and after arsenite addition (100 µM). C) Carbon content of the different strains analyzed before and after arsenite addition (100 µM). Data are means ± SD from 2 biological replicates with 3 technical replicates at each point. 1496 |PLANT PHYSIOLOGY 2024: 195; 1491–1505 Domínguez-Lobo et al. Downloaded from https://academic.oup.com/plphys/article/195/2/1491/7611675 by Biblioteca de la Universidad de Sevilla user on 05 June 2024 were used as controls under the same conditions. Samples for the metabolic analysis were taken after 0, 3, 8, and 24 h from the induction of cfrA with arsenite. In a WT genetic background for glycogen synthesis, cfrA overexpression caused the accumulation of carbon storage compounds such as sucrose or GG, in addition to the already described glycogen production (Fig. 6A). However, when cfrA was induced in a ΔglgC strain, virtually all carbon compounds upstream of the 3PG experienced a substantial increase early with the addition of the inductor (Fig. 6B). The accumulation of all these sugars phosphate (hexoses, pentoses, trioses, etc.) took place rapidly, reaching high levels in almost all cases already 3 h after the addition of the cfrA inducer. This is consistent with the rapid accumulation observed for CfrA protein (Fig. 1C). Especially important was the rise in fructose-1,6-bisphosphate, more than 20 times, 3 h after the addition of arsenite, and 30 times higher after 24 h of induction in ΔglgC/Pars-cfrA strain. In a similar range, sedoheptulose-7-phosphate, erythrose-4-phosphate (E4P), fructose-6-phosphate, glucose-6-phosphate, dihydroxyacetone phosphate, and 6-phosphogluconate also increased at 3 and 24 h of the induction. None of these compounds accumulated appreciably in the Pars-cfrA strain compared to the control WT strain after adding Figure 5. Time course LC-MS analysis of selected metabolites linked to the OAC cycle in the Pars-cfrA strain and the WT strain as a control. The quantification of these compounds over time (0, 3, 8, and 24 h) after the addition of arsenite (50 µM) is shown. Each bar represents the metabolite level at a certain time, determined from 3 independent experiments with 3 technical replicates at each point. The error bars represent the SD of the combined data. The values are in nanograms per OD 750 . Carbon metabolism in cfrA overexpressors PLANT PHYSIOLOGY 2024: 195; 1491–1505 |1497 Downloaded from https://academic.oup.com/plphys/article/195/2/1491/7611675 by Biblioteca de la Universidad de Sevilla user on 05 June 2024 A B Figure 6. Time course LC-MS analysis of metabolites derived from the CO 2 fixation product 3PG, as a function of cfrA expression. The PGAM reaction, negatively regulated by CfrA, is marked with a cross. A) Carbon storage compounds that accumulated substantially after cfrA induction in a WT background for ADP-glucose synthesis are marked in red. The quantification of these compounds over time (0, 3, 8, and 24 h) after the addition of arsenite (50 µM) is shown. B) The reaction catalyzed by AGP, blocked in strains ΔglgC and ΔglgC/Pars-cfrA, is marked with a cross. The carbon compounds that strongly accumulated after the expression of cfrA in a strain blocked in the synthesis of ADP-glucose are marked in red. In A) and B), each bar represents the metabolite level at a certain time, determined from 3 independent experiments with 3 technical replicates at each point. The error bars represent the SD of the combined data. The values are in nanograms per optical density at 750 nm. The results of all analyzed carbohydrates are shown in Supplementary Fig. S3. 1,3BPG, 1,3-bisphosphoglycerate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; 6PG, 6-phospho- gluconate; DHAP, dihydroxyacetone phosphate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; GAP, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate; R5P, ribose-5- phosphate; Ru5P, ribulose-5-phosphate; RuBP, ribulose-1,5-bisphosphate; S7P, sedoheptulose-7-phosphate; SBP, sedoheptulose-1,7-bisphosphate; Xu5P, xylulose-5-phosphate. 1498 |PLANT PHYSIOLOGY 2024: 195; 1491–1505 Domínguez-Lobo et al. Downloaded from https://academic.oup.com/plphys/article/195/2/1491/7611675 by Biblioteca de la Universidad de Sevilla user on 05 June 2024 arsenite. Since the photosynthetic assimilation of carbon is a main source of intermediates for amino acid synthesis, we also analyzed amino acid levels as a function of cfrA expression. In the ΔglgC/Pars-cfrA strain, unable to synthesize glycogen, the induction of cfrA led to a large increase in some amino acids (Fig. 7). Thus, serine and glycine, whose synthesis derives from 3PG, increased 15 and 9 times, respectively, after cfrA induction. It is interesting to note that 3PG could be accumulated by the action of CfrA on the PGAM enzyme and that in fact increased in the ΔglgC/Pars-cfrA strain after the addition of arsenite (Fig. 6B). Nonetheless, the other amino acids that increased substantially in this strain: alanine (16 times), whose synthesis takes place directly from pyruvate by the action of alanine dehydrogenase, valine (10 times), leucine (8 times), and isoleucine (5 times) originate from pyruvate, a metabolite that is found downstream of the action of PGAM in the glycolytic pathway. None of these amino acids undergo appreciable changes after the addition of arsenite in the Pars-cfrA strain (Fig. 7). The full spectra of the metabolites analyzed by LC-MS are shown in Supplementary Figs. S3 to S6. An increase of around 2-fold was observed in the pool of tryptophan and tyrosine of strain ΔglgC/Pars-cfrA after the addition of arsenite (Supplementary Fig. S4), which is in agreement with the increase in E4P, a precursor of their synthesis (Fig. 6B). It is worth noting that glutamate, the more abundant amino acid in Synechocystis, exhibited low fluctuation in the analyzed conditions. As expected, the ΔglgC strains do not accumulate ADP-glucose; however, there are no substantial differences in the accumulation of UDP-glucose between the different genetic backgrounds analyzed (Supplementary Fig. S6). Discussion Here, we describe the metabolic effects of cfrA overexpression in different strains, based on their ability to synthesize ADP-glucose and its derivatives, mainly glycogen. The results show that the overexpression of cfrA in cells unable to Figure 7. Time course LC-MS analysis of amino acids and pyruvate as a function of cfrA expression, depicted in a schematic representation of metabolic pathways from 3PG, including glycolysis, the TCA cycle, and the GS-GOGAT cycle. The PGAM reaction, negatively regulated by CfrA, is marked with a cross. Amino acids, derived from 3PG and pyruvate, that strongly accumulated after the expression of cfrA, in a strain blocked in the synthesis of ADP-glucose are marked in red. Each bar represents the metabolite level at a certain time, determined from 3 independent experiments with 3 technical replicates at each point. The error bars represent the SD of the combined data. The results of all analyzed amino acids are shown in Supplementary Fig. S4 and other intermediaries in Supplementary Fig. S5. 2-OG, 2-oxoglutarate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; AcAcetyl-CoA, aceto-acetyl-CoA; Ac-CoA, acetyl-CoA; MA, malate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PHB, polyhydroxybutyrate; Pyr, pyruvate: Suc, succinate. Carbon metabolism in cfrA overexpressors PLANT PHYSIOLOGY 2024: 195; 1491–1505 |1499 Downloaded from https://academic.oup.com/plphys/article/195/2/1491/7611675 by Biblioteca de la Universidad de Sevilla user on 05 June 2024