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The adc1 knockout with proC overexpression in Synechocystis sp. PCC 6803 induces a diversion of acetyl-CoA to produce more polyhydroxybutyrate

Abstract

Background

Lack of nutrients, in particular nitrogen and phosphorus, has been known in the field to sense glutamate production via 2-oxoglutarate and subsequently accelerate carbon storage, including glycogen and polyhydroxybutyrate (PHB), in cyanobacteria, but a few studies have focused on arginine catabolism. In this study, we first time demonstrated that gene manipulation on proC and adc1, related to proline and polyamine syntheses in arginine catabolism, had a significant impact on enhanced PHB production during late growth phase and nutrient-modified conditions. We constructed Synechocystis sp. PCC 6803 with an overexpressing proC gene, encoding Δ1pyrroline-5-carboxylate reductase in proline production, and adc1 disruption resulted in lower polyamine synthesis.

Results

Three engineered Synechocystis sp. PCC 6803 strains, including a ProC-overexpressing strain (OXP), adc1 mutant, and an OXP strain lacking the adc1 gene (OXP/Δadc1), certainly increased the PHB accumulation under nitrogen and phosphorus deficiency. The possible advantages of single proC overexpression include improved PHB and glycogen storage in late phase of growth and long-term stress situations. However, on day 7 of treatment, the synergistic impact created by OXP/Δadc1 increased PHB synthesis by approximately 48.9% of dry cell weight, resulting in a shorter response to nutrient stress than the OXP strain. Notably, changes in proline and glutamate contents in engineered strains, in particular OXP and OXP/Δadc1, not only partially balanced the intracellular C/N metabolism but also helped cells acclimate under nitrogen (N) and phosphorus (P) stress with higher chlorophyll a content in comparison with wild-type control.

Conclusions

In Synechocystis sp. PCC 6803, overexpression of proC resulted in a striking signal to PHB and glycogen accumulation after prolonged nutrient deprivation. When combined with the adc1 disruption, there was a notable increase in PHB production, particularly in situations where there was a strong C supply and a lack of N and P.

Introduction

The cumulative harm caused by pollution and inadequate resource and land management of our remaining natural resources is a major connected element of many global concerns. Microalgae, including cyanobacteria, have the ability to address some of these issues by decreasing aquatic pollutants and offering a sustainable supply of biomass for product development, as evidenced by the emerging uses of microalgal biotechnology assisting the United Nations’ Sustainable Development Goals (SDGs) [1, 2]. The microalgal biomass possesses a wide variety of primary and secondary metabolites that are increasingly being acknowledged for their importance in the production of novel products and biotechnological applications. These valuable products, which can be produced directly from CO2 via photosynthesis, include pigments such as carotenoids, and chlorophylls, carbon storages such as glycogen and polyhydroxybutyrate (PHB), as well as macromolecular compounds such as proteins, carbohydrates, and lipids [3,4,5,6,7,8,9]. There are several strategies for boosting algal biomass in order to achieve the lower carbon restriction that limits the yield of biofuel and bioproducts, including nutritional adjustment, and gene manipulation by genetic and metabolic engineering. The increased CO2 fixation capacity, such as RuBisCO gene overexpression, and glucose utilization, has driven cyanobacteria to have better growth and photosynthesis [6, 10,11,12], and be able to produce more PHB and lipids [4]. Under nitrogen or phosphorus deficiency, most cyanobacteria preferentially store carbon sources in the forms of glycogen and PHB, which are a consequence of the 2-oxoglutarate balance for carbon/nitrogen control [13,14,15]. In Fig. 1, the arginine catabolism flux associated with the polyamine synthesis, proline-glutamate reaction, and GS/GOGAT pathway [16, 17]. Previous studies found that a transposon-mutated Synechocystis sp. PCC 6803 significantly enhanced PHB synthesis while lacking the proA gene encoding gamma-glutamyl phosphate reductase. This mutant also had decreased proline reduction and increased glutamate accumulation [18]. Furthermore, the arginine–ornithine to proline–glutamate reaction was primarily driven by the deletion of the adc1 gene, which encodes arginine decarboxylase involved in polyamine biosynthesis, in Synechocystis sp. PCC 6803. This undoubtedly boosted PHB production, although the precise mechanism is still unknown [5]. Notably, in Synechocystis sp. PCC 6803, during nitrogen shortage, the activation of an OmpR-type response regulator (Rre37) may stimulate the metabolic flux from glycogen to PHB as well as the hybrid TCA cycle and arginine–ornithine cycle [9]. In cyanobacteria, glutamate can be synthesized through two alternative systems including GS/GOGAT pathway, and another catalyzed by glutamate dehydrogenase (GDH) [19, 20]. The GS/GOGAT pathway is the main ammonium assimilation system in Synechocystis 6803, while GDH encoded by the gdhA gene is regulated by the late stage of growth [21, 22] and when energy supply is limited in Escherichia coli [23]. On the other hand, when acetyl-CoA flow is driven to PHB accumulation, it is mostly directed to the TCA cycle and fatty acid synthesis from pyruvate and acetate, except for nutritional constraints (Fig. 1). Enzymes which involved in PHB biosynthesis are β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and heterodimeric PHB synthase (phaE and phaC), respectively [24]. Instead of producing new CO2 fixation, the cyanobacteria that were starved of nutrients favored producing PHB from internally stored carbon storage, such as glycogen [8, 25]. In this study, to supply more glutamate to TCA cycle, we overexpressed the proC gene, encoding Δ1pyrroline-5-carboxylate reductase (Fig. 1), in Synechocystis sp. PCC6803 wild type and Δadc1 mutant strains. Two engineered strains included a Synechocystis sp. PCC6803 overexpressing proC gene or OXP, and Synechocystis sp. PCC6803 overexpressing proC gene with a knockout of adc1 gene in polyamine synthesis or OXP/Δadc1 strains. Both engineered strains certainly accumulated higher PHB content, particularly in a nitrogen and phosphorus-deprived BG11 medium with acetate supplementation (BG11-N-P + A). It is important to highlight that, particularly in the presence of BG11-N-P + A, the acetyl-CoA flow was mainly diverted to the PHB biosynthetic pathway.

Fig. 1
figure 1

Overview of the polyamine-proline-glutamate pathways connected to the tricarboxylic acid (TCA) cycle and related biosynthetic pathways of polyhydroxybutyrate (PHB), and glycogen in cyanobacterium Synechocystis sp. PCC 6803. Abbreviations of genes are: acc, a multisubunit acetyl-CoA carboxylase; ach, acetyl-CoA hydrolase; ackA, acetate kinase; acs, acetyl-CoA synthase; adc, arginine decarboxylase; argD, N-acetylornithine aminotransferase; gad, glutamate decarboxylase; gdhA, glutamate dehydrogenase; glgC, ADP-glucose pyrophosphorylase; glgX, glycogen debranching enzyme; gltA, citrate synthase; plsX, fatty acid/phospholipid synthesis protein; phaA, β-ketothiolase; phaB, acetoacetyl-CoA reductase; phaC and phaE, the heterodimeric PHB synthase; proA, gamma-glutamyl phosphate reductase; proC, Δ1pyrroline-5-carboxylate reductase; pta, phosphotransacetylase; putA, proline oxidase; speB1, arginase; speB2, agmatinase. Abbreviations of intermediates are: FASII, fatty acid synthesis type II; GABA, gamma-aminobutyric acid; GOGAT, glutamate synthase; G6P, glucose-6-phosphate; GS, glutamine synthetase; 3-PGA, 3-phosphoglycerate; PHB, polyhydroxybutyrate

Results

Overexpression of native proC gene in Synechocystis sp. PCC 6803 wild-type and mutant strains

Initially, we constructed four engineered Synechocystis sp. PCC 6803 strains, including wild-type control (WTc), Δadc1 mutant control (Δadc1c), OXP, and OXP/Δadc1 by double homologous recombination (Table 1, Fig. 2A). For the WTc and Δadc1c strains, they were created by replacing the psbA2 gene with a CmR cassette in the genomes of Synechocystis sp. PCC 6803 WT and Δadc1 mutant, respectively (Fig. 2A). To create a recombinant plasmid pEERM_proC (Table 1), a native proC (or slr0661) gene fragment with a size of 1.0 kb was ligated between flanking regions of the psbA2 gene of the pEERM vector and the upstream region of CmR cassette (Fig. 2A). Next, all overexpressing strains were verified by PCR using specific pairs of primers (Additional file 1: Table S1) for their complete segregation and gene location. To confirm the complete segregation, PCR products with Up_psbA2-F and Dw_psbA2-R primers confirmed the correct size of 3.2 kb, in OXP and OXP/Δadc1 strains (Fig. 2B.1 and B.2, respectively), while there were 2.3 kb in WT and Δadc1 strains, and 2.2 kb in WTc and Δadc1c strains. PCR products with ProC-F and CmR-R primers confirmed the correct size of 1.9 kb in OXP and OXP/Δadc1 strains (Fig. 2C1 and C2, respectively), compared with no band in WT, WTc, Δadc1 and Δadc1c strains. In addition, ProC gene overexpression was verified by RT-PCR data in all engineered strains (Fig. 2D).

Table 1 Strains and plasmids used in this study
Fig. 2
figure 2

Genomic maps and transcript levels of Synechocystis sp. PCC 6803 strains. The four constructed strains are Synechocystis sp. PCC 6803 wild-type control (WTc), Synechocystis sp. PCC 6803 lacking adc1 gene (Δadc1c), Synechocystis sp. PCC 6803 overexpressing proC gene (OXP), and Δadc1 mutant overexpressing proC gene (OXP/Δadc1). PCR analysis employing two pairs of specific primers (Supplementary information Table S1) was used to confirm the accurate integration and placement of each gene fragment into the Synechocystis genome. (A) The double homologous recombination of both CmR gene occurred between the conserved sequences of psbA2 gene in WTc and Δadc1c, and a proC:CmR fragment occurred between the conserved sequences of psbA2 gene in OXP and OXP/Δadc1 strains when compared to WT. (B) For PCR products using UP_psbA2-F and Dw_psbA2-R primers, (B.1) For OXP strain, Lane M: GeneRuler DNA ladder, Lanes OX1, OX2, and OX3: three clones no. 1–3 containing a 3.2 kb fragment of Up_psbA2-proC-CmR-Dw_psbA2, Lanes WT and WTc: negative controls of a 2.4 kb fragment in WT and a 2.2 kb fragment in WTc, respectively. (B.2) For OXP/Δadc1 strain, Lane M: GeneRuler DNA ladder, Lanes OX1 and OX2: two clones no. 1 and 2 containing a 3.2 kb fragment of Up_psbA2-proC-CmR-Dw_psbA2, Lanes Δadc1 and Δadc1c: negative controls of a 2.4 kb fragment in Δadc1 and a 2.2 kb fragment in Δadc1c, respectively. (C) For PCR products using ProC-F and CmR-R primers, (C.1) For OXP strain, Lane M: GeneRuler DNA ladder, Lanes OX1, OX2, and OX3: three clones no. 1–3 containing a 1.9 kb fragment of proC-CmR, Lanes WT and WTc: negative controls (no band) using WT and WTc as template, respectively. (C.2) For OXP/Δadc1 strain, Lane M: GeneRuler DNA ladder, Lanes Δadc1 and Δadc1c: negative controls (no band) using Δadc1 and Δadc1c as template, respectively, Lanes OX1 and OX2: two clones no. 1 and 2 containing a 1.9 kb fragment of proC-CmR. (D) Transcript levels of proC gene determined by RT-PCR using RT-ProC-F and RT-ProC-R primers (Additional file 1: Table S1) in WT, WTc, Δadc1, Δadc1c, and two overexpressing strains, including OXP and OXP/Δadc1. The 0.8% agarose gel electrophoresis of PCR products was performed from cells grown for 6 days in normal BG11 medium. The 16s rRNA was used as reference. The cropped gels (in D) were taken from the original images of RT-PCR products on agarose gels as shown in Supplementary information Figure S1

Growth, intracellular pigment contents, oxygen evolution rates, and metabolite accumulation under normal growth condition

We found a slight increase in cell growth of the Δadc1c and OXP/Δadc1 strains in comparison with the WTc and OXP strains (Fig. 3A). All strains had comparable amounts of chlorophyll a; however, proC overexpression in OXP and OXP/Δadc1 had an impact on the decrease of carotenoids (Fig. 3B, C). In comparison to WTc, all engineered strains showed lower rates of oxygen evolution (Fig. 3D). On the other hand, as anticipated, total polyamines (PAs) in both bound and free forms declined in the Δadc1c and OXP/Δadc1 strains, but the OXP strain showed a minor decrease in total PAs as compared to the WTc strain (Fig. 3E). It was found that bound PAs were the main decrease when the adc1 gene was disrupted. On day 7 under normal growth condition, the proline levels of OXP and OXP/Δadc1 strains were found to be much higher, but the Δadc1c strain has the lowest proline content (Fig. 3F). Moreover, the WTc strain exhibited significant glutamate content that was almost ten times greater than proline under normal growth conditions (Fig. 3G). All mutant strains, especially the OXP/Δadc1 strain, had a greater increase in glutamate content than the WTc. Similarly, on day 7 of culture, the GABA level in WTc was higher than the proline content but somewhat lower than the glutamate content under normal condition (Fig. 3H). The GABA content of all engineered strains, especially OXP/Δadc1, was lower than that of the WTc strain. Glutamate appeared to be the preferred compound that Synechocystis cells accumulated, followed by GABA and proline. On the other hand, cells substantially produced a low level of PHB by about 4 – 23% w/DCW under normal growth condition (Fig. 3I). When compared to the WTc, the PHB quantity in the OXP and OXP/Δadc1 strains appeared to be larger. In order to adapt to the nutrient-modified medium, cells growing on day 11, which represents the late-log phase of cell growth with the maximum level of PHB accumulation, were subsequently selected.

Fig. 3
figure 3

Growth curve (A), chlorophyll a content (B), carotenoid content (C), oxygen evolution rate (D), contents of polyamines (E), proline (F), glutamate (G), GABA (H), and PHB (I) of WTc, Δadc1c, OXP, and OXP/Δadc1 Synechocystis sp. PCC 6803 strains. In (AC), and (I), cells grown in BG11 medium for 16 days. In (DH), cells were grown in normal BG11 medium for 7 days, and harvested for metabolite contents. The error bars represent standard deviations of means (mean ± S.D., n = 3). In (DI), the statistical difference of the results between those values of WTc and that engineered strain is indicated by an asterisk at *P < 0.05

Growth, intracellular pigment contents, and metabolite accumulation under nutrient-modified conditions

All Synechocystis strains were grown in normal BG11 medium for 11 days before starting the adaptation phase (Fig. 4). Both two nutrient-modified media, including BG11 lacking nitrogen and phosphorus (BG11-N-P) and BG11-N-P medium with acetate addition (BG11-N-P + A), caused a certain reduction in growth (Fig. 4A–C) and intracellular contents of chlorophyll a and carotenoids (Fig. 4D–I). It is worth noting that all engineered strains had a slightly higher level of cell growth under the BG11-N-P + A condition, in particular Δadc1c (Fig. 4C). In addition, the proC-overexpressing strains, including OXP and OXP/Δadc1, contained a higher accumulation of chlorophyll a than WTc under the BG11-N-P + A condition (Fig. 4F).

Fig. 4
figure 4

Growth curve (AC), chlorophyll a content (DF), and carotenoid content (GI) of Synechocystis WTc, Δadc1c, OXP, and OXP/Δadc1 strains adapted in normal BG11 medium, BG11 medium with N and P deprivation (BG11-N-P), and BG11-N-P supplemented with 4%(w/v) acetate (BG11-N-P + A) medium for 11 days. The error bars represent standard deviations of means (mean ± S.D., n = 3). The statistical difference of the results between those values of WTc and that engineered strain is indicated by an asterisk at *P < 0.05

For the main carbon storages of glycogen and polyhydroxybutyrate (PHB) (Fig. 5), glycogen was markedly accumulated rather than PHB under normal growth condition, in particular for a longer period at days 9–11 of cultivation (Fig. 5A and D). The OXP strain contained the highest level of glycogen, up to 30–49% of dry cell weight, among other strains, during 9–11 days under normal BG11 condition (Fig. 5D). Both BG11-N-P and BG11-N-P + A conditions promoted the specific induction of PHB synthesis in all strains, whereas cells comparatively reduced the quantity of glycogen compared to those under normal condition (Fig. 5B–F). Remarkably, on day 7 of the adaptation phase of both BG11-N-P and BG11-N-P + A media, the OXP/Δadc1 strain accumulated the greatest amount of PHB, with around 39.2 and 48.9%w/DCW, respectively (Fig. 5B, C). Moreover, in Fig. 6, there was a 2.7-fold increase in PHB in the OXP/Δadc1 strain compared to WTc at day 7 under a BG11-N-P + A condition. Interestingly, after adapting to both BG11-N-P and BG11-N-P + A media, the PHB accumulation of the OXP strain was later driven to reach its maximum level on day 9. On the other hand, it was anticipated that these BG11-N-P and BG11-N-P + A conditions would result in a reduction in polyamines in all strains, particularly OXP/Δadc1 strain (Table 2). It was evident from Fig. 6 that the OXP/Δadc1 strain has decreased by 0.8 fold in comparison to WTc (Fig. 6). Regarding the proline-glutamate-GABA pathway, glutamate production predominated under typical BG11 condition, particularly in OXP and OXP/Δadc1 strains, followed by GABA and proline (Table 2). The proline content was presumably increased by BG11-N-P and BG11-N-P + A conditions, based on the greater fold change compared to WTc in OXP and OXP/Δadc1 strains (Fig. 6). Glutamate accumulation was reduced (Table 2), but in the modified strains, specifically the OXP strain, it increased by more than 5—7 times in both BG11-N-P and BG11-N-P + A conditions, compared to the WTc (Fig. 6). Moreover, GABA accumulation was mostly decreased under nutrient-modified conditions.

Fig. 5
figure 5

Contents of PHB (AC) and glycogen (DF) of Synechocystis WTc, Δadc1c, OXP, and OXP/Δadc1 strains adapted in normal BG11 medium, BG11 medium with N and P deprivation (BG11-N-P), and BG11-N-P supplemented with 4%(w/v) acetate (BG11-N-P + A) medium for 11 days. The error bars represent standard deviations of means (mean ± S.D., n = 3). An asterisk (*P < 0.05) denotes the statistical difference in results between those WTc values and that engineered strain at each day

Fig. 6
figure 6

Fold changes of obtained results of metabolite products in three engineered strains compared with those in Synechocystis WTc after adapting cells in BG11, BG11-N-P, and BG11-N-P + A for 7 days. In each box, the number represents the fold change of that value of each engineered strain under each stress condition divided by that value of WTc. The statistical difference in the data between those values of WT and the engineered strain is represented by an asterisk at *P < 0.05

Table 2 Contents of some metabolites related to polyamine-proline-glutamate pathway. Cells were grown in various media for 7 days (means ± S.D., n = 3). The statistical difference in the data between the values of WT and the engineered strain is represented by an asterisk at * p < 0.05

We also stained cells adapted under the BG11-N-P + A condition for 7 days with Nile red dye and visualized them under fluorescent microscopy (Fig. 7A). When compared to other strains, the OXP/Δadc1 strain manifestly exhibited a high abundance of PHB granules in entire cells. Furthermore, RT-PCR was conducted to measure the transcript levels of 15 different genes (Fig. 7B, C). Both in OXP and OXP/Δadc1 under normal and BG11-N-P + A conditions, the proC transcript level increased. It is noteworthy that OXP and OXP/Δadc1 strains likewise exhibited elevated putA transcript levels, encoding proline oxidase. Furthermore, BG11-N-P + A condition increased the transcript levels of the gdhA and gad genes, encoding glutamate dehydrogenase and glutamate decarboxylase, respectively, with the exception of the Δadc1c strain. The transcript levels of the acs, ach, and ackA genes, encoding acetyl-CoA synthase, acetyl-CoA hydrolase, and acetate kinase, respectively, in acetate metabolism, were increased by the acetate supplementation in BG11-N-P medium. Remarkably, all strains under the BG11-N-P + A condition showed an increase in the transcript level of the gltA gene, encoding citrate synthase in a first step of the TCA cycle, when compared to the normal BG11 condition. On the other hand, although the BG11-N-P + A condition raised the quantity of the accA transcript, encoding acetyl-CoA carboxylase subunit A in fatty acid synthesis, relative to the normal condition, there was a low alteration in the plsX transcript level, encoding fatty acid/phospholipid synthesis protein. Strikingly, transcript levels of all pha genes, including phaA, phaB, phaC, and phaE, were upregulated by BG11-N-P + A condition. The reduction of glgX transcript amount, encoding glycogen debranching enzyme in glycogen degradation, was induced by the BG11-N-P + A condition rather than the normal BG11 control.

Fig. 7
figure 7

The Nile red stained PHB granules (A), relative transcript levels (B), and their band intensity ratios of gene/16s (C) of genes involved in PHB synthesis, glycogen degradation, proline-glutamate conversion, and neighboring pathways in Synechocystis WTc, Δadc1c, OXP, and OXP/Δadc1 strains under BG11-N-P + A condition at day 7 of treatment. The 16s rRNA was used as reference control

Discussion

The disruption of the polyamine synthetic adc1 gene in Synechocystis sp. PCC 6803 was initially discovered in a previous study [5], but the metabolic regulation remained unclear. In this work, we highlight the remarkable finding that higher PHB synthesis (up to 48.9% of dry cell weight) was caused by enhanced metabolic flux from arginine to proline and glutamate, which is closely related to nutrient stress. The introduction of the native proC gene, encoding pyrroline-5-carboxylate reductase of proline synthesis, in Synechocystis sp. PCC 6803 wild type (WT) and adc1 mutant (Δadc1) was constructed, thereby creating OXP and OXP/Δadc1 strains, respectively. The proC mutant of Synechocystis sp. PCC 6803 was previously shown to produce less proline, but a putA mutant that lacks the enzyme proline oxidase, which breaks down proline to glutamate, nonetheless accumulated a high amount of proline metabolites without producing any glutamate [16]. Enhanced proline accumulation is indicated in response to environmental stress [27,28,29]. In plants, the stress response of proline accumulation was controversial depending on different species and organisms; maize seedlings had an increased proline production in response to nitrogen and phosphorus deficiency [30], while French bean (Phaseolus vulgaris L cv Strike) plants showed a decline in proline accumulation under nitrogen-deprived condition [31], as well as a low proline level in Arabidopsis thaliana growing under nitrogen-limiting condition [32]. In our study, regarding BG11-N-P and BG11-N-P + A conditions, we found a minor alteration in proline accumulation in WTc as compared to BG11 control (Table 2). It is worthy to note that the adc1 knockout with a decreased polyamine also contained lower proline content than the WTc, except for the BG11-N-P condition. Our results also indicated that glutamate accumulation in all strains was dramatically decreased in response to nitrogen and phosphorus deprivation in comparison to the WTc. Nevertheless, glutamate content increased more than twofold in all engineered strains as compared to WTc results (Fig. 8), particularly in OXP and OXP/Δadc1 strains. Amidst the deficiency of nitrogen and phosphorus, glutamate might have taken up a rapid key role in the metabolism of amino acids through pathways including the GS/GOGAT pathway, multispecific aminotransferases, GABA synthesis, and reversible conversions to proline and arginine [33,34,35,36,37]. Interestingly, the transcript level of the gdhA gene, encoding glutamate dehydrogenase (GDH), was upregulated only in the OXP strain which contained the highest level of glutamate under the BG11-N-P + A condition (Fig. 7B, C), with 1.77-fold increase compared to WTc (Fig. 8). Our finding demonstrated that the proC overexpression and adc1 disruption in Synechocystis (OXP/Δadc1) had noted results in higher proline and/or glutamate contents in comparison with WTc, which partially alleviated cells under nitrogen and phosphorus deficiency, as evidenced by the increased accumulation of chlorophyll a, although the stress effect of nutrient deprivation still existed.

Fig. 8
figure 8

Fold changes of obtained results of gene transcript levels and metabolite contents in three engineered strains compared with those in Synechocystis WTc after adapting cells in BG11-N-P + A for 7 days. In each box and graph, the number and bar graph represents the fold change of that value of each engineered strain divided by that value of WTc, respectively

In addition, we demonstrated that, under typical BG11 condition, when cells reached the late phase of growth on day 11, they accumulated more glycogen (Fig. 5D), in particular the OXP strain with 49%w/DCW, and had less PHB (Fig. 5A). The growth phase of cyanobacterial cells, in which the levels of ATP and ADP are elevated during the lag phase and then dropped during the log phase, is directly attributed to the energy charge. The improved metabolism and storage of glycogen significantly contribute to maintaining energy homeostasis [38]. Moreover, the nitrogen and phosphorus deficiency certainly accelerated the glycogen accumulation within 3 days of the adaptation phase (Fig. 5E), as well as PHB production (Fig. 5B). The impact of glycogen breakdown on PHB synthesis during nitrogen deprivation has been previously reported [8]. Subsequently, in order to improve the acetyl-CoA substrate for PHB synthesis, we added carbon source, herein acetate, to the BG11-N-P medium. On day 7 of the adaptation phase, there was a noticeable increase in PHB accumulation in OXP/Δadc1 of around 48.9% w/DCW (Fig. 5C). The increased acetate utilization in the OXP/Δadc1 strain in comparison to other strains confirmed this finding (Additional file 1: Fig. S2). Our results indicated that, with the exception of the OXP strain, Synechocystis cells favored using exogenous acetate to acetyl-CoA over glycogen breakdown, as demonstrated by a reduced amount of glgX transcript under BG11-N-P + A condition (Fig. 7B, C). Regarding acetate metabolism, cells acclimated to a BG11-N-P medium containing acetate exhibited significantly higher levels of the transcripts ackA and acs, encoding acetyl-CoA synthase and acetate kinase, respectively. This is consistent with their enhanced fold change, as noted in Fig. 8. It is worth noting that the OXP strain also had a higher ackA transcript level than that under normal BG11 medium (Fig. 7B), but it showed a decreased fold change when compared to WTc (Figs. 7C and 8). According to these data, the OXP strain may have utilized acetate less than other strains, which may have contributed to its reduced PHB contents on day 7 under BG11-N-P + A treatment compared to the Δadc1c and OXP/Δadc1 strains. This finding was confirmed by higher amount of acetate remaining in the medium during treatment with the OXP strain than other strains (Additional file 1: Fig. S2). As demonstrated earlier by the acs mutant, which did not use the external acetate in medium, it is crucial to stress that the Acs enzyme functions as the primary metabolic route for acetate absorption in Synechocystis sp. PCC 6803 [39]. Remarkably, we suggested that the flow of acetyl-CoA metabolite to citrate in the TCA cycle in all strains was induced by the BG11-N-P + A condition, as supported by the upregulated transcript level of gltA gene, encoding citrate synthase (Fig. 7B, C). Nonetheless, it is crucial to note that the gltA transcript levels in the engineered strains, including OXP, adc1c, and OXP/Δadc1, were lower than the WTc (Fig. 8). This result indicated that the lowered flow of acetyl-CoA to the TCA cycle in engineered strains in comparison to WTc substantially contributed to driving acetyl-CoA to other flux directions, such as PHB biosynthetic pathway and fatty acid synthesis. Then, we postulated that the increased levels of proline and glutamate in the engineered strains OXP, Δadc1c, and OXP/Δadc1 were substantially related to the flow of acetyl-CoA to the TCA cycle and a conversion between 2-oxoglutarate and glutamate. Our results have not provided the TCA cycle’s metabolites; further identification of pertinent metabolites or the application of integrative bioinformatic approaches might increase our knowledge of the actual mechanism. For PHB synthesis, it is important to note that all pha genes in the PHB synthetic pathway were increased in their transcript amounts under the BG11-N-P + A condition, in particular the phaC and phaE genes, in comparison to the normal BG11 condition (Fig. 7B, C). Nonetheless, our findings demonstrate a strong correlation between the increased amounts of phaA and phaB transcripts and the improved synthesis of PHB in the modified strains (OXP, Δadc1c, and OXP/Δadc1) (Fig. 8). This was in line with a previous study in Synechocystis sp. PCC6803, where increased PHB synthesis was associated with overexpression of the phaAB gene rather than phaEC overexpression during nitrogen deprivation [40]. On the other hand, the acetyl-CoA direction to fatty acid synthesis was also induced by the BG11-N-P + A condition due to the high upregulation of the accA transcript level and a slight induction of the plsX transcript (Fig. 7B, C). This could imply that acetate addition contributes to lipid production in cyanobacteria [40, 41]. According to Ref. [42], cyanobacterial cells grown in high C/low N conditions functioned by preventing the inhibitory interaction of PII protein with ACCase, while cells grown in high N/low C conditions could enhance the PII-ACCase interaction, leading to an inhibition of the ACCase enzyme.

Conclusions

The nitrogen and phosphorus-deprived condition efficiently induced the accumulation of glycogen and PHB in Synechocystis sp. PCC 6803. In this study, higher PHB production was attained in three modified Synechocystis sp. PCC6803 strains, including Δadc1c, OXP, and OXP/Δadc1, under the nutrient-deprived treatments, in particular nitrogen and phosphorus-deprived BG11 medium with acetate addition (BG11-N-P + A). The proC overexpression and adc1 knockout in Synechocystis apparently induced the changes in proline and glutamate contents inside the cells, which partially alleviated cells under nitrogen and phosphorus deprivation. However, the acetate addition, enhancing acetyl-CoA metabolite, significantly boosted the PHB and glycogen storage. These genetically modified strains of Synechocystisadc1c, OXP, and OXP/Δadc1) might serve as practicable cell factories for biotechnological applications including biomaterials and biofuels.

Materials and Methods

Construction of proC-overexpressing Synechocystis sp. PCC 6803

First, the recombinant plasmid pEERM_ProC was constructed (Table 1), which was naturally transformed into Synechocystis sp. PCC 6803 wild type (WT) and Δadc1 mutant (obtained from [5]), thereby generating a proC-overexpressing Synechocystis sp. PCC 6803 (OXP), and an OXP lacking adc1 gene (OXP/Δadc1), respectively. The pEERM_proC plasmid was constructed by ligating the proC gene fragment amplified by PCR using a pair of ProC-F and ProC-R primers, as shown in Additional file 1: Table S1, in between the SpeI and PstI restriction sites in the pEERM vector [26]. The correct recombinant plasmid pEERM_proC was transformed into WT and Δadc1 mutant cells by natural transformation to create OXP and OXP/Δadc1 strains, respectively. In addition, we also constructed the Synechocystis sp. PCC 6803 wild-type control (WTc) and the Δadc1 mutant control (Δadc1c) by transforming the empty pEERM vector into WT and Δadc1 cells, represented as Synechocystis WT or Δadc1 mutant containing the CmR cassette gene (Fig. 2A). For host cell suspension preparation, the host cells (WT or Δadc1) were cultures in BG11 medium until OD730 reaching about 0.3–0.5. Then, 10 mL of cell culture was harvested by centrifugation at 5500 rpm (3505 × g), 25 °C for 10 min, and cell pellets were resuspended in 500 μL of new BG11 medium. Next step, the host cell suspension was mixed with 10 μL of recombinant plasmid solution, and incubated that mixture overnight in the culture room with continuous light illumination at 40–50 μE/m2/s, at 28–30 °C. Then, the sample mixture was spread on a BG11 agar plate containing 10 μg/mL of chloramphenicol, and incubated in the culture room for 2–3 weeks until survived colonies occurred on plate. Each single colony was picked and streaked on a new BG11 agar plate containing higher concentrations of chloramphenicol (20 and 30 μg/mL), and incubated under same condition until transformant colonies appeared. The obtained transformants were confirmed for gene size, location, and segregation by PCR method using many specific pairs of primers (Additional file 1: Table S1).

Strains and culture conditions

Synechocystis sp. PCC 6803 wild type (WT), derived from the Berkeley strain 6803 from fresh water in California, USA [43], Synechocystis lacking adc1 gene (Δadc1), and all engineered strains (WTc, Δadc1c, OXP, and OXP/Δadc1) were grown in normal BG11 medium for 16 days. The culture room, set for normal growth condition, was performed at 28–30 °C, with a continuous white light illumination by 40–50 μE/m2/s intensity. The cell culture flasks with the initial cell density at 730 nm (OD730) of about 0.05 were placed on the rotary shaker at 160 rpm speed. Cell growth was measured at OD730 by spectrophotometer. For nutrient-deprived conditions, all Synechocystis strains were initially grown in normal BG11 medium until late-log phase of cell growth before treating them with nutrient-derived media under the same growth condition for 11 days. There were two modified media including a BG11 medium without nitrogen (N) and phosphorus (P) (or BG11-N-P), and a BG11-N-P medium with 0.4%(w/v) acetate (A) addition (or BG11-N-P + A). For BG11-N-P medium, it was a BG11 medium lacking NaNO3 with KCl added in place of KH2PO4, and FeSO4 added in place of ferric ammonium citrate in equimolar concentrations [5]. In addition, the initial OD730 of cell culture under nutrient-modified conditions was adjusted to about 0.2. Acetate concentration in medium was determined according to the method of Ref. [44].

Determinations of intracellular pigments and oxygen evolution rate

Cell culture (1 mL) was harvested by centrifugation at 12,000 rpm (14,383 × g) for 10 min. The intracellular pigments including chlorophyll a and carotenoids in cell pellets were extracted by N,N-dimethylformamide (DMF) (1 mL), vortexed and incubated under darkness for 10 min. After centrifugation at the same speed, the absorbance of the yellowish supernatant was spectrophotometrically measured at 461, 625, and 664 nm. The contents of chlorophyll a and carotenoids were calculated according to Refs. [45, 46]. For oxygen evolution rate, cell culture (10 mL) was harvested by centrifugation at 5500 rpm (3505 × g), 25 °C for 10 min. Cell pellets were resuspended in new BG11 medium (1 mL) and incubated under darkness for 30 min before measuring the oxygen evolution. Saturated light source was employed at 25 °C using Clark-type oxygen electrode (Hansatech instruments Ltd., King’s Lynn, UK). The unit of oxygen evolution rate was addressed as μmol O2/mg chlorophyll a/h [5].

Total RNAs extraction and reverse transcription-polymerase reaction (RT-PCR)

Total RNAs were extracted from Synechocystis cells using the TRIzol® Reagent (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA). The purified RNAs (1 μg) were converted to cDNA by reverse transcription using ReverTra ACE® qPCR RT Master Mix Kit (TOYOBO Co., Ltd., Osaka, Japan). This obtained cDNA was subsequently used as a template for PCR with different pairs of primers (Additional file 1: Table S1). The PCR conditions were performed by initial denaturing at 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, annealing temperature of each gene (Additional file 1: Table S1) for 30 s, and 72 °C for 35 s, followed by a final extension at 72 °C for 5 min. For 16s rRNA reference, the PCR condition was the same, but there was 19 cycles instead. Prior to initiating the experiment, the optimum cycle for all genes was determined. Those bands were not saturated; instead, they were in the appropriate cycle. The PCR products were checked by 1.5% (w/v) agarose gel electrophoresis. Quantification of band intensity was detected by Syngene® Gel Documentation (Syngene, Frederick, MD, USA).

HPLC analysis of PHB contents and Nile red staining

Cell cultures (50 mL) were harvested by centrifugation at 5500 rpm (3505 × g) for 10 min. To prepare sample for HPLC detection, cell pellets were hydrolyzed by boiling for 60 min with 98%(v/v) sulfuric acid (800 μL) and 20 mg/mL adipic acid (100 μL), an internal standard [5]. After that, the hydrolyzed sample was filtered using a 0.45 μm polypropylene membrane filter which was further detected by HPLC instrument (Shimadzu HPLC LGE System, Kyoto, Japan) using a carbon-18 column, Inert sustain 3-μm (GL Science, Tokyo, Japan), with a UV detector at 210 nm. The running buffer consisted of 30% (v/v) acetonitrile dissolved in 10 mM KH2PO4 (pH 7.4), with a flow rate of 1.0 mL/min. Authentic commercial PHB was used as standard, which was prepared as same as the samples. For dry cell weight (DCW), it was determined by incubating cell pellets in an oven at 80 °C for 16–18 h, until obtaining the constant weight.

For Nile red staining, cell culture (1 mL) was harvested by centrifugation at 5500 rpm (3505 × g) for 10 min. Cell pellets were resuspended in Nile red staining solution (3 μL). Then, the addition of normal saline (0.9%, w/v, 100 μL) was conducted and incubated overnight under darkness [5, 40]. To visualize the stained cells, fluorescent microscope (Carl Zeiss, Oberkochen, Jena, Germany) was applied using a filter cup with 535 excitation wavelength, at a magnification of 100x.

Extraction and determination of glycogen content

Harvested cell pellets from liquid culture (15–30 mL) were extracted by alkaline hydrolysis [47, 48]. The 30% potassium hydroxide (KOH) solution (400 μL) was mixed with cell pellets, and boiled for 1 h. After centrifugation at 12,000 rpm (14,383 \(\times \) g), 4 °C for 10 min, the supernatant was transferred to a new tube and mixed with 900 μL of cold absolute ethanol before incubating at − 20 °C overnight to precipitate glycogen. Next step, the sample mixture was centrifuged at 12,000 rpm (14,383 \(\times \)g), 4 °C for 30 min to obtain glycogen pellets which were subsequently dried at 60 °C overnight. Glycogen pellets were dissolved in 1 mL of 10% H2SO4. Then, dissolved sample (0.2 mL) was mixed with 10% H2SO4 (0.2 mL) and anthrone reagent (0.8 mL) before boiling for 10 min. After cooling down the samples to room temperature, the absorbance of samples was measured at 625 nm by spectrophotometer (modified from [4, 7]). A commercial oyster glycogen was used as the standard which was prepared as similar as the sample. The unit of glycogen content was %w/DCW.

Extraction and determination of polyamine content

Total polyamines were extracted from Synechocystis cells with 5% cold HClO4 (modified from [49, 50]. After extraction by 5% cold HClO4 for 1 h on ice, the extracted samples were centrifuged at 12,000 rpm (14,838 \(\times \) g) for 10 min. The supernatant and pellet fractions were represented as the fraction containing free and bound forms of polyamines, respectively. Both fractions were used to derivatize and quantify the total polyamines. For the derivatization, it was performed with benzoyl chloride using 1,6-hexanediamine as an internal standard. 1 mL of 2 M NaOH was mixed with 500 µL of HClO4 extract and 10 µL of benzoyl chloride. After vigorously mixing, the mixture was incubated for 20 min at room temperature. To terminate the reaction, saturated NaCl solution (2 mL) was added. The benzoyl polyamines were subsequently extracted by cold diethyl ether (2 mL). In addition, the ether phase (1 mL) was evaporated to dryness, and redissolved in methanol (1 mL). Authentic polyamine standards were prepared as similar as the samples. The polyamine content was detected by high-performance liquid chromatography (HPLC; Shimadzu HPLC LGE System, Kyoto, Japan) with inertsil®ODS-3 C-18 reverse phase column (5 μm; 4.6 × 150 mm) with UV–Vis detector at 254 nm. The mobile phase was a gradient of 60–100% methanol with a flow rate of 0.5 mL/min.

Quantification of proline, glutamate, and GABA contents

HPLC detection of amino acids, including proline, glutamate, and GABA, was performed using o-phthalaldehyde (OPA) and 9-Fluorenylmethyl chloroformate (FMOC) derivatives (modified from [51, 52]). Cell pellets obtained from cell culture (50 mL) were washed and resuspended in 10 mM potassium phosphate citrate buffer (pH 7.6). Cell suspensions were homogenized using SONOPLUS Ultrasonic homogenizer (BANDELIN electronic GmbH & Co., Berlin, Germany). The supernatant was collected after centrifugation at 12,000 rpm (14,838 \(\times \) g) for 10 min, and concentrated by a Centrivap concentrator (Labconco Corporation, MO, USA). The concentrated sample was further extracted with 600 μL of a mixture of water:chloroform:methanol (3:5:12,v/v/v), followed by 300 μL of chloroform and 450 μL of distilled water before centrifugation again at 5500 rpm (3505 × g), 4 ºC, for 10 min. The upper fraction of water–methanol phase was collected, and evaporated before redissolving in 200 μL of 0.1 N HCl. The sample solution was filtered through a 0.45 μm membrane filter, and then diluted (1:4,v/v) with internal standard solution of norvaline and sarcosine (62.5 mM in 0.1 M HCl). Then, this mixture was again filtered through a 0.45 μm membrane filter before detecting by HPLC with UV–VIS detector (Shimadzu HPLC LGE System, Kyoto, Japan) using 4.6 × 150 mm, 3.5 μm Agilent Zorbax Eclipse AAA analytical column and 4.6 × 12.5 mm, 5.0 μm guard column (Agilent Technologies, CA, USA). For the mobile phase, eluent A was 40 mM Na2HPO4, pH 7.8, and eluent B was acetonitrile:methanol:water (45:45:10, v/v/v), with a flow rate of 2 mL/min. The OPA- and FMOC-derivatized amino acids were monitored at 338 and 262 nm, respectively. The unit of amino acid content was nmol/mg protein.

Availability of data and materials

Data generated and analyzed during this study are included in the published article.

Abbreviations

ADC:

Arginine decarboxylase

Arg:

Arginine

Car:

Carotenoids

Chl a :

Chlorophyll a

DCW:

Dry cell weight

DMF:

N,N-Dimethylformamide

FMOC:

9-Fluorenylmethyl chloroformate

GABA:

Gamma-aminobutyric acid

h:

Hour

μg:

Microgram

mL:

Milliliter

min:

Minute

nm:

Nanometer

OD:

Optical density

OPA:

O-Phthalaldehyde

PCR:

Polymerase chain reaction

PHB:

Polyhydroxybutyrate

rpm:

Revolutions per minute

s:

Seconds

WT:

Wild type

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Acknowledgements

We gratefully thank Professor Peter Lindblad, Microbial Chemistry, Department of Chemistry–Ångström, Uppsala University, for providing the expression vector pEERM for our work.

Funding

This research was supported by the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) to S.U. and S.J. This Research is also funded by Thailand Science research and Innovation Fund Chulalongkorn University (CU_FRB65_hea(66)_129_23_59) to SJ.

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SU responsible for study conception, experimenter, data collection and analysis, manuscript preparation. SJ study conception, supervision, and design, critical revision and manuscript writing, and final approval of the manuscript. All the authors read and approved the final manuscript.

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Correspondence to Saowarath Jantaro.

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Supplementary Information

Additional file 1: Table S1.

Primers used in this study. Figure S1. Agarose gel electrophoresis of RT-PCR products of proC transcript in Synechocystis sp. PCC 6803 strains grown under normal BG11 condition for 6 days, shown in Figure 2C. The 16s rRNA transcript was used as the reference (a size of 521 bp). ProC transcript size of 315 bp. Figure S2. Acetate concentration in BG11-N-P+A medium during adaptation phase of all strains. Cells were treated in BG11-N-P+A medium for 11 days. Medium was sampled at days 0, 1, 3, 5, 7, 9, and 11 for determining acetate concentration (according to the method of Hutchens and Kass, 1949). The error bars represent standard deviations of means (mean ± S.D., n = 3).

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Utharn, S., Jantaro, S. The adc1 knockout with proC overexpression in Synechocystis sp. PCC 6803 induces a diversion of acetyl-CoA to produce more polyhydroxybutyrate. Biotechnol Biofuels 17, 6 (2024). https://doi.org/10.1186/s13068-024-02458-9

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