Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.algal.2024.103526

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Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

DOI： 10.1007/s11103-023-01401-0

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Cyanobacteria are oxygen-evolving photosynthetic prokaryotes that affect the global carbon and nitrogen turnover. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a model cyanobacterium that has been widely studied and can utilize and uptake various nitrogen sources and amino acids from the outer environment and media. l-arginine is a nitrogen-rich amino acid used as a nitrogen reservoir in Synechocystis 6803, and its biosynthesis is strictly regulated by feedback inhibition. Argininosuccinate synthetase (ArgG; EC 6.3.4.5) is the rate-limiting enzyme in arginine biosynthesis and catalyzes the condensation of citrulline and aspartate using ATP to produce argininosuccinate, which is converted to l-arginine and fumarate through argininosuccinate lyase (ArgH). We performed a biochemical analysis of Synechocystis 6803 ArgG (SyArgG) and obtained a Synechocystis 6803 mutant overexpressing SyArgG and ArgH of Synechocystis 6803 (SyArgH). The specific activity of SyArgG was lower than that of other arginine biosynthesis enzymes and SyArgG was inhibited by arginine, especially among amino acids and organic acids. Both arginine biosynthesis enzyme-overexpressing strains grew faster than the wild-type Synechocystis 6803. Based on previous reports and our results, we suggest that SyArgG is the rate-limiting enzyme in the arginine biosynthesis pathway in cyanobacteria and that arginine biosynthesis enzymes are similarly regulated by arginine in this cyanobacterium. Our results contribute to elucidating the regulation of arginine biosynthesis during nitrogen metabolism.

DOI： 10.1007/s11103-024-01416-1

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Fumarase is an enzyme catalyzing reversible reaction between fumarate and L-malate in the citric acid cycle. Fumarase is used in the industrial production of L-malate, and its immobilization is required for reuse of the fumarases to reduce the cost. Accordingly, understanding the properties of immobilized fumarase is crucial, and several groups report on the storage stability and kinetic parameters of immobilized fumarase. Here we have immobilized fumarase from the thermophilic red alga Cyanidioschyzon merolae (CmFUM) on ceramic beads and investigated its biochemical and physical properties. CmFUM demonstrated sufficient stability and reusability for industry use after immobilization. Notably, the thermostability was dramatically enhanced through immobilization. The Km value and kcat of immobilized CmFUM for fumarate were 1.7 mM and 22.7 s-1 respectively. The Km value for fumarate was lower than that of other reported immobilized fumarases, indicating a high substrate affinity of immobilized CmFUM. Furthermore, the enhanced stability resulting from immobilization partially compensated for the decrease in activity. The high affinity towards fumarate and good thermostability of immobilized CmFUM revealed in this study are advantageous traits for improving enzyme-mediated isomer-specific L-malate production.

DOI： 10.2323/jgam.2024.02.003

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Cyanobacteria serve as useful hosts in the production of substances to support a low-carbon society. Specifically, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) can produce organic acids, such as acetate, lactate, and succinate, as well as hydrogen, under dark, anaerobic conditions. The efficient production of these compounds appears to be closely linked to the regulation of intracellular redox balance. Notably, alterations in intracellular redox balance have been believed to influence the production of organic acids and hydrogen. To achieve these alterations, genetic manipulations involved overexpressing malate dehydrogenase (MDH), knocking out d-lactate dehydrogenase (DDH), or knocking out acetate kinase (AK), which subsequently modified the quantities and ratios of organic acids and hydrogen under dark, anaerobic conditions. Furthermore, the mutants generated displayed changes in the oxidation of reducing powers and the nicotinamide adenine dinucleotide hydrogen (NADH)/NAD+ ratio when compared to the parental wild-type strain. These findings strongly suggest that intracellular redox balance, especially the NADH/NAD+ ratio, plays a pivotal role in the production of organic acids and hydrogen in Synechocystis 6803.

DOI： 10.3389/fmicb.2023.1332449

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

Abstract

Cyanobacteria have been promoted as a biomass resource that can contribute to carbon neutrality. Synechocystis sp. PCC 6803 is a model cyanobacterium that is widely used in various studies. NADP+ and NAD+ are electron receptors involved in energy metabolism. The NADP+/NAD+ ratio in Synechocystis sp. PCC 6803 is markedly higher than that in the heterotrophic bacterium Escherichia coli. In Synechocystis sp. PCC 6803, NADP+ primarily functions as an electron receptor during the light reaction of photosynthesis, and NADP+ biosynthesis is essential for photoautotrophic growth. Generally, the regulatory enzyme of NADP+ biosynthesis is NAD kinase, which catalyzes the phosphorylation of NAD + . However, a previous study suggested that the regulation of another enzyme contributes to NADP+ biosynthesis in Synechocystis sp. PCC 6803 under photoautotrophic conditions. l-Aspartate oxidase is the first enzyme in NAD(P)+ biosynthesis. We biochemically characterized Synechocystis sp. PCC 6803 l-aspartate oxidase and determined the phenotype of a Synechocystis sp. PCC 6803 mutant overexpressing l-aspartate oxidase. The catalytic efficiency of l-aspartate oxidase from Synechocystis sp. PCC 6803 was lower than that of l-aspartate oxidases and NAD kinases from other organisms. l-Aspartate oxidase activity was affected by different metabolites, such as NADP+ and ATP. The l-aspartate oxidase-overexpressing strain grew faster compared with the wild-type strain under photoautotrophic conditions. The l-aspartate oxidase-overexpressing strain accumulated NADP+ under photoautotrophic conditions. These results indicate that the regulation of l-aspartate oxidase contributes to NADP+ biosynthesis in Synechocystis sp. PCC 6803 under photoautotrophic conditions. These findings provide insight into the regulatory mechanism of cyanobacterial NADP+ biosynthesis.

DOI： 10.1093/plphys/kiad580

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DOI： 10.1016/j.algal.2023.103088

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Euglena gracilis (E. gracilis) is a unicellular microalga with various applications in medicine, agriculture, aquaculture, health supplement, and jet fuel production. Euglena possibly solves population growth and exhaustion of fossil resources. Efficient cell harvesting is needed for the industry, and the gravity sedimentation method is low cost and does not require any equipment, although it has low efficiency. This study showed that the gravity sedimentation of E. gracilis cells is improved by cultivation in the presence of ethanol (EtOH). The gravity sedimentation of E. gracilis cells cultivated under 0.5% or 1.0% EtOH conditions was faster than that cultivated without EtOH. The mean calculated cell diameter was also found to be largest in cells cultivated under 0.5% or 1.0% EtOH conditions compared to that in cells cultivated without EtOH. Intracellular paramylon content, cell shapes, and motility differed between cells cultivated under 0.5% or 1.0% EtOH conditions and in the absence of EtOH. The results suggest that E. gracilis cultivation with EtOH leads to increased cell productivity, paramylon production, and efficient cell harvesting. KEY POINTS: • Euglena gracilis is an edible microalga producing value-added metabolites. • Ethanol addition upregulates E. gracilis growth and paramylon accumulation. • Gravity sedimentation is accelerated by ethanol-grown E. gracilis cells.

DOI： 10.1007/s00253-023-12476-6

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Citrate synthase (CS) catalyzes the reaction that produces citrate and CoA from oxaloacetate and acetyl-CoA in the tricarboxylic acid (TCA) cycle. All TCA cycle enzymes are localized to the mitochondria in the model organism, the red alga Cyanidioschyzon merolae. The biochemical properties of CS have been studied in some eukaryotes, but the biochemical properties of CS in algae, including C. merolae, have not been studied. We then performed the biochemical analysis of CS from C. merolae mitochondria (CmCS4). The results showed that the kcat/Km of CmCS4 for oxaloacetate and acetyl-CoA were higher than those of the cyanobacteria, such as Synechocystis sp. PCC 6803, Microcystis aeruginosa PCC 7806 and Anabaena sp. PCC 7120. Monovalent and divalent cations inhibited CmCS4, and in the presence of KCl, the Km of CmCS4 for oxaloacetate and acetyl-CoA was higher in the presence of MgCl2, the Km of CmCS4 for oxaloacetate and acetyl-CoA was higher and kcat lower. However, in the presence of KCl and MgCl2, the kcat/Km of CmCS4 was higher than those of the three cyanobacteria species. The high catalytic efficiency of CmCS4 for oxaloacetate and acetyl-CoA may be a factor in the increased carbon flow into the TCA cycle in C. merolae.

DOI： 10.1007/s11103-023-01335-7

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The four-carbon (C4) dicarboxylic acids, fumarate, malate, and succinate, are the most valuable targets that must be exploited for CO2-based chemical production in the move to a sustainable low-carbon future. Cyanobacteria excrete high amounts of C4 dicarboxylic acids through glycogen fermentation in a dark anoxic environment. The enhancement of metabolic flux in the reductive TCA branch in the Cyanobacterium Synechocystis sp. PCC6803 is a key issue in the C4 dicarboxylic acid production. To improve metabolic flux through the anaplerotic pathway, we have created the recombinant strain PCCK, which expresses foreign ATP-forming phosphoenolpyruvate carboxykinase (PEPck) concurrent with intrinsic phosphoenolpyruvate carboxylase (Ppc) overexpression. Expression of PEPck concurrent with Ppc led to an increase in C4 dicarboxylic acids by autofermentation. Metabolome analysis revealed that PEPck contributed to an increase in carbon flux from hexose and pentose phosphates into the TCA reductive branch. To enhance the metabolic flux in the reductive TCA branch, we examined the effect of corn-steep liquor (CSL) as a nutritional supplement on C4 dicarboxylic acid production. Surprisingly, the addition of sterilized CSL enhanced the malate production in the PCCK strain. Thereafter, the malate and fumarate excreted by the PCCK strain are converted into succinate by the CSL-settling microorganisms. Finally, high-density cultivation of cells lacking the acetate kinase gene showed the highest production of malate and fumarate (3.2 and 2.4 g/L with sterilized CSL) and succinate (5.7 g/L with non-sterile CSL) after 72 h cultivation. The present microbial community engineering is useful for succinate production by one-pot fermentation under dark anoxic conditions.

DOI： 10.1021/acssynbio.2c00379

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Oxygenic photoautotrophic bacteria, cyanobacteria, have the tricarboxylic acid (TCA) cycle, and metabolite production using the cyanobacterial TCA cycle has been spotlighted recently. The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (Synechocystis 6803) has been used in various studies on the cyanobacterial TCA cycle. Malate oxidation in the TCA cycle is generally catalyzed by malate dehydrogenase (MDH). However, Synechocystis 6803 MDH (SyMDH) is less active than MDHs from other organisms. Additionally, SyMDH uses only NAD+ as a coenzyme, unlike other TCA cycle enzymes from Synechocystis 6803 that use NADP+. These results suggest that MDH rarely catalyzes malate oxidation in the cyanobacterial TCA cycle. Another enzyme catalyzing malate oxidation is malic enzyme (ME). We clarified which enzyme oxidizes malate that originates from the cyanobacterial TCA cycle using analyses focusing on ME and MDH. In contrast to SyMDH, Synechocystis 6803 ME (SyME) showed high activity when NADP+ was used as a coenzyme. Unlike the Synechocystis 6803 mutant lacking SyMDH, the mutant lacking SyME accumulated malate in the cells. ME was more highly preserved in the cyanobacterial genomes than MDH. These results indicate that ME mainly oxidizes malate that originates from the cyanobacterial TCA cycle (named the ME-dependent TCA cycle). The ME-dependent TCA cycle generates NADPH, not NADH. This is consistent with previous reports that NADPH is an electron carrier in the cyanobacterial respiratory chain. Our finding suggests the diversity of enzymes involved in the TCA cycle in the organisms, and analyses such as those performed in this study are necessary to determine the enzymes. IMPORTANCE Oxygenic photoautotrophic bacteria, namely, cyanobacteria, have the tricarboxylic acid (TCA) cycle. Recently, metabolite production using the cyanobacterial TCA cycle has been well studied. To enhance the production volume of metabolites, understanding the biochemical properties of the cyanobacterial TCA cycle is required. Generally, malate dehydrogenase oxidizes malate in the TCA cycle. However, cyanobacterial malate dehydrogenase shows low activity and does not use NADP+ as a coenzyme, unlike other cyanobacterial TCA cycle enzymes. Our analyses revealed that another malate oxidation enzyme, the malic enzyme, mainly oxidizes malate that originates from the cyanobacterial TCA cycle. These findings provide better insights into metabolite production using the cyanobacterial TCA cycle. Furthermore, our findings suggest that the enzymes related to the TCA cycle vary from organism to organism and emphasize the importance of analyses to identify the enzymes such as those performed in this study.

DOI： 10.1128/mbio.02187-22

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β-Amylase hydrolyzes polysaccharides, such as starch, into maltose. It is used as an industrial enzyme in the production of food and pharmaceuticals. The eukaryotic red alga Cyanidioschyzon merolae is a unicellular alga that grows at an optimum pH of 2.0-3.0 and an optimum temperature of 40-50 °C. By focusing on the thermostability and acid resistance of the proteins of C. merolae, we investigated the properties of β-amylase from C. merolae (hereafter CmBAM) and explored the possibility of using CmBAM as an industrial enzyme. CmBAM showed the highest activity at 47 °C and pH 6.0. CmBAM had a relatively higher specificity for amylose as a substrate than for starch. Immobilization of CmBAM on a silica gel carrier improved storage stability and thermostability, allowing the enzyme to be reused. The optimum temperature and pH of CmBAM were comparable to those of existing β-amylases from barley and wheat. C. merolae does not use amylose, but CmBAM has a substrate specificity for both amylose and amylopectin but not for glycogen. Among the several β-amylases reported, CmBAM was unique, with a higher specificity for amylose than for starch. The high specificity of CmBAM for amylose suggests that isoamylase and pullulanase, which cleave the α-1,6 bonds of starch, may act together in vivo. Compared with several reported immobilized plant-derived β-amylases, immobilized CmBAM was comparable to β-amylase, with the highest reusability and the third-highest storage stability at 30 days of storage. In addition, immobilized CmBAM has improved thermostability by 15-20 °C, which can lead to wider applications and easier handling.

DOI： 10.1021/acsomega.2c03315

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KEY MESSAGE: This study revealed different catalytic efficiencies of cyanobacterial argininosuccinate lyases in non-nitrogen-fixing and nitrogen-fixing cyanobacteria, demonstrating that L-arginine inhibition of L-argininosuccinate lyase is conserved among enzymes of three cyanobacterial orders. Arginine is a nitrogen-rich amino acid that uses a nitrogen reservoir, and its biosynthesis is strictly controlled by feedback inhibition. Argininosuccinate lyase (EC 4.3.2.1) is the final enzyme in arginine biosynthesis that catalyzes the conversion of argininosuccinate to L-arginine and fumarate. Cyanobacteria synthesize intracellular cyanophycin, which is a nitrogen reservoir composed of aspartate and arginine. Arginine is an important source of nitrogen for cyanobacteria. We expressed and purified argininosuccinate lyases, ArgHs, from Synechocystis sp. PCC 6803, Nostoc sp. PCC 7120, and Arthrospira platensis NIES-39. The catalytic efficiency of the Nostoc sp. PCC 7120 ArgH was 2.8-fold higher than those of Synechocystis sp. PCC 6803 and Arthrospira platensis NIES-39. All three ArgHs were inhibited in the presence of arginine, and their inhibitory effects were lowered at pH 7.0, compared to those at pH 8.0. These results indicate that arginine inhibition of ArgH is widely conserved among the three cyanobacterial orders. The current results demonstrate the conserved regulation of enzymes in the cyanobacterial aspartase/fumarase superfamily.

DOI： 10.1007/s11103-022-01280-x

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DOI： 10.1111/tpj.15687

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A unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses a unique tricarboxylic acid (TCA) cycle, wherein the intracellular citrate levels are approximately 1.5-10 times higher than the levels of other TCA cycle metabolite. Aconitase catalyses the reversible isomerisation of citrate and isocitrate. Herein, we biochemically analysed Synechocystis sp. PCC 6803 aconitase (SyAcnB), using citrate and isocitrate as the substrates. We observed that the activity of SyAcnB for citrate was highest at pH 7.7 and 45 °C and for isocitrate at pH 8.0 and 53 °C. The Km value of SyAcnB for citrate was higher than that for isocitrate under the same conditions. The Km value of SyAcnB for isocitrate was 3.6-fold higher than the reported Km values of isocitrate dehydrogenase for isocitrate. Therefore, we suggest that citrate accumulation depends on the enzyme kinetics of SyAcnB, and 2-oxoglutarate production depends on the chemical equilibrium in this cyanobacterium.

DOI： 10.1038/s41598-021-96432-2

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Succinate, fumarate, and malate are valuable four-carbon (C4) dicarboxylic acids used for producing plastics and food additives. C4 dicarboxylic acid is biologically produced by heterotrophic organisms. However, current biological production requires organic carbon sources that compete with food uses. Herein, we report C4 dicarboxylic acid production from CO2 using metabolically engineered Synechocystis sp. PCC 6803. Overexpression of citH, encoding malate dehydrogenase (MDH), resulted in the enhanced production of succinate, fumarate, and malate. citH overexpression increased the reductive branch of the open cyanobacterial tricarboxylic acid (TCA) cycle flux. Furthermore, product stripping by medium exchanges increased the C4 dicarboxylic acid levels; product inhibition and acidification of the media were the limiting factors for succinate production. Our results demonstrate that MDH is a key regulator that activates the reductive branch of the open cyanobacterial TCA cycle. The study findings suggest that cyanobacteria can act as a biocatalyst for converting CO2 to carboxylic acids.

DOI： 10.1016/j.ymben.2021.03.007

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The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways in nature. Oxygenic photoautotrophic bacteria, cyanobacteria, have an unusual TCA cycle. The TCA cycle in cyanobacteria contains two unique enzymes that are not part of the TCA cycle in other organisms. In recent years, sustainable metabolite production from carbon dioxide using cyanobacteria has been looked at as a means to reduce the environmental burden of this gas. Among cyanobacteria, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) is an optimal host for sustainable metabolite production. Recently, metabolite production using the TCA cycle in Synechocystis 6803 has been carried out. Previous studies revealed that the branch point of the oxidative and reductive TCA cycles, oxaloacetate metabolism, plays a key role in metabolite production. However, the biochemical mechanisms regulating oxaloacetate metabolism in Synechocystis 6803 are poorly understood. Concentrations of oxaloacetate in Synechocystis 6803 are extremely low, such that in vivo analysis of oxaloacetate metabolism does not seem realistic. Therefore, using purified enzymes, we reconstituted oxaloacetate metabolism in Synechocystis 6803 in vitro to reveal the regulatory mechanisms involved. Reconstitution of oxaloacetate metabolism revealed that pH, Mg2+ and phosphoenolpyruvate are important factors affecting the conversion of oxaloacetate in the TCA cycle. Biochemical analyses of the enzymes involved in oxaloacetate metabolism in this and previous studies revealed the biochemical mechanisms underlying the effects of these factors on oxaloacetate conversion. In addition, we clarified the function of two l-malate dehydrogenase isozymes in oxaloacetate metabolism. These findings serve as a basis for various applications of the cyanobacterial TCA cycle.

DOI： 10.1111/tpj.15120

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C-Phycocyanin (PC) is a protein used commercially as a natural blue pigment produced by cyanobacteria, cryptophytes, and rhodophytes. Although it is industrially synthesized from the cyanobacterium Arthrospira platensis, PC requires high levels of energy for its extraction, which involves freezing of cells. However, as a protein, PC is easily denatured at extreme temperatures. In this study, we extracted PC from the red alga Cyanidioschyzon merolae, denoted as CmPC, and found that this protein was tolerant to high temperatures and acidic pH. CmPC was extracted by suspending cells in water mixed with various salts and organic acids without freeze-drying or freeze-thaw. The stability of CmPC varied with salt concentration and was destabilized by organic acids. Our results indicate that C. merolae is a potential candidate for PC production with thermotolerant properties.

DOI： 10.1016/j.jbiosc.2020.09.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

DOI： 10.1016/j.algal.2020.102084

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In photosynthetic microorganisms, cell cycle progression depends on day and night cycles; however, how cell division is regulated in response to these environmental changes is poorly understood. RpaA has been implicated in the signal output from both circadian clocks and light/dark conditions in the unicellular spherical-celled cyanobacterium Synechocystis sp. PCC 6803. In the present study, we investigated the involvement of a two-component response regulator RpaA in cell division regulation. Firstly, we examined the effects of rpaA overexpression on cell morphology and the expression levels of cell division genes. We observed an increase in the volume of non-dividing cells and a high proportion of dividing cells in rpaA-overexpressing strains by light microscopy. The expression levels of selected cell division-related genes were higher in the rpaA-overexpressing strain than in the wild type, including minD of the Min system; cdv3 and zipN, which encode two divisome components; and murB, murC, and pbp2, which are involved in peptidoglycan (PG) synthesis. Moreover, in the rpaA-overexpressing strain, the outer membrane and cell wall PG layer were not smooth, and the outer membrane was not clearly visible by transmission electron microscopy. These results demonstrated that rpaA overexpression causes an impaired cell division, which is accompanied by transcriptional activation of cell division genes and morphological changes in the PG layer and outer membrane.

DOI： 10.2323/jgam.2020.01.004

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Synechocystis sp. PCC 6803, a cyanobacterium widely used for basic research, is often cultivated in a synthetic medium, BG-11, in the presence of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) or 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid buffer. Owing to the high cost of HEPES buffer (96.9% of the total cost of BG-11 medium), the biotechnological application of BG-11 is limited. In this study, we cultured Synechocystis sp. PCC 6803 cells in BG-11 medium without HEPES buffer and examined the effects on the primary metabolism. Synechocystis sp. PCC 6803 cells could grow in BG-11 medium without HEPES buffer after adjusting for nitrogen sources and light intensity; the production rate reached 0.54 g cell dry weight·L-1 ·day-1 , exceeding that of commercial cyanobacteria and Synechocystis sp. PCC 6803 cells cultivated under other conditions. The exclusion of HEPES buffer markedly altered the metabolites in the central carbon metabolism; particularly, the levels of compatible solutes, such as sucrose, glucosylglycerol, and glutamate were increased. Although the accumulation of sucrose and glucosylglycerol under high salt conditions is antagonistic to each other, these metabolites accumulated simultaneously in cells grown in the cost-effective medium. Because these metabolites are used in industrial feedstocks, our results reveal the importance of medium composition for the production of metabolites using cyanobacteria.

DOI： 10.1002/bit.27324

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Metabolite production from carbon dioxide using sugar catabolism in cyanobacteria has been in the spotlight recently. Synechocystis sp. PCC 6803 (Synechocystis 6803) is the most studied cyanobacterium for metabolite production. Previous in vivo analyses revealed that the oxidative pentose phosphate (OPP) pathway is at the core of sugar catabolism in Synechocystis 6803. However, the biochemical regulation of the OPP pathway enzymes in Synechocystis 6803 remains unknown. Therefore, we characterized a key enzyme of the OPP pathway, glucose-6-phosphate dehydrogenase (G6PDH), and related enzymes from Synechocystis 6803. Synechocystis 6803 G6PDH was inhibited by citrate in the oxidative tricarboxylic acid (TCA) cycle. Citrate has not been reported as an inhibitor of G6PDH before. Similarly, 6-phosphogluconate dehydrogenase, the other enzyme from Synechocystis 6803 that catalyzes the NADPH-generating reaction in the OPP pathway, was inhibited by citrate. To understand the physiological significance of this inhibition, we characterized succinic semialdehyde dehydrogenase (SSADH) from Synechocystis 6803 (SySSADH), which catalyzes one of the NAD(P)H generating reactions in the oxidative TCA cycle. Similar to isocitrate dehydrogenase from Synechocystis 6803, SySSADH specifically catalyzed the NADPH-generating reaction and was not inhibited by citrate. The activity of SySSADH was lower than that of other bacterial SSADHs. Previous and this studies revealed that unlike the OPP pathway, the oxidative TCA cycle is a pathway with low efficiency in NADPH generation in Synechocystis 6803. It has, thus, been suggested that to avoid NADPH overproduction, the OPP pathway dehydrogenase activity is repressed when the flow of the oxidative TCA cycle increases in Synechocystis 6803.

DOI： 10.1042/BCJ20200038

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d-Lactate is one of the most valuable compounds for manufacturing biobased polymers. Here, we have investigated the significance of endogenous malate dehydrogenase (decarboxylating) (malic enzyme, ME), which catalyzes the oxidative decarboxylation of malate to pyruvate, in d-lactate biosynthesis in the cyanobacterium Synechocystis sp. PCC6803. d-Lactate levels were increased by 2-fold in ME-overexpressing strains, while levels in ME-deficient strains were almost equivalent to those in the host strain. Dynamic metabolomics revealed that overexpression of ME led to increased turnover rates in malate and pyruvate metabolism; in contrast, deletion of ME resulted in increased pool sizes of glycolytic intermediates, probably due to sequential feedback inhibition, initially triggered by malate accumulation. Finally, both the loss of the acetate kinase gene and overexpression of endogenous d-lactate dehydrogenase, concurrent with ME overexpression, resulted in the highest production of d-lactate (26.6 g/L) with an initial cell concentration of 75 g-DCW/L after 72 h fermentation.

DOI： 10.1021/acssynbio.9b00281

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Fumarases (Fums) catalyze the reversible reaction converting fumarate to l-malate. There are two kinds of Fums: Class І and ІІ. Thermostable Class ІІ Fums, from mesophilic microorganisms, are utilized for industrial l-malate production. However, the low thermostability of these Fums is a limitation in industrial l-malate production. Therefore, an alternative Class ІІ Fum that shows high activity and thermostability is required to overcome this drawback. Thermophilic microalgae and cyanobacteria can use carbon dioxide as a carbon source and are easy to cultivate. Among them, Cyanidioschyzon merolae and Thermosynechococcus elongatus are model organisms to study cell biology and structural biology, respectively. We biochemically analyzed Class ІІ Fums from C. merolae (CmFUM) and T. elongatus (TeFum). Both CmFUM and TeFum preferentially catalyzed fumarate hydration. The catalytic activity of CmFUM for fumarate hydration in the optimum conditions (52°C and pH 7.5) is higher compared to those of Class ІІ Fums from other organisms and TeFum. Thermostability tests of CmFUM revealed that CmFUM showed higher thermostability than those of Class ІІ Fums from other microorganisms. The yield of l-malate obtained from fumarate hydration catalyzed by CmFUM was 75-81%. In summary, CmFum has suitable properties for efficient l-malate production.

DOI： 10.3389/fmicb.2020.560894

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DOI： 10.1016/j.algal.2019.101565

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The tricarboxylic acid cycle produces NADH for oxidative phosphorylation and fumarase [EC 4.2.1.2] is a critical enzyme in this cycle, catalysing the reversible conversion of fumarate and L-malate. Fumarase is applied to industrial L-malate production as a biocatalyst. L-malate is used in a wide range of industries such as food and beverage, pharmacy chemistry. Although the biochemical properties of fumarases have been studied in many organisms, they have not been investigated in cyanobacteria. In this study, the optimum pH and temperature of Synechocystis 6803 fumarase C (SyFumC) were 7.5 and 30 °C, respectively. The Km of SyFumC for L-malate was higher than for fumarate. Furthermore, SyFumC activity was strongly inhibited by citrate and succinate, consistent with fumarases in other organisms. Substitution of alanine by glutamate at position 314 of SyFumC changed the kcat for fumarate and L-malate. In addition, the inhibitory effects of citrate and succinate on SyFumC activity were alleviated. Phylogenetic analysis revealed cyanobacterial fumarase clades divided in non-nitrogen-fixing cyanobacteria and nitrogen-fixing cyanobacteria. SyFumC was thus biochemically characterised, including identification of an amino acid residue important for substrate affinity and enzymatic activity.

DOI： 10.1038/s41598-019-47025-7

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Citrate synthase (CS, EC 2.3.3.1) catalyses the initial reaction of the tricarboxylic acid (TCA) cycle. Although CSs from heterotrophic bacteria have been extensively studied, cyanobacterial CSs are not well-understood. Cyanobacteria can produce various metabolites from carbon dioxide. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a cyanobacterium used to synthesize metabolites through metabolic engineering techniques. The production of acetyl-CoA-derived metabolites in Synechocystis 6803 has been widely examined. However, the biochemical mechanisms of reactions involving acetyl-CoA in Synechocystis 6803 are poorly understood. We characterised the CS from Synechocystis 6803 (SyCS) and compared its characteristics with other bacterial CSs. SyCS catalysed only the generation of citrate, and did not catalyse the cleavage of citrate. It is suggested that SyCS is not related to the reductive TCA cycle. The substrate affinity and turnover number of SyCS were lower than those of CSs from heterotrophic bacteria. SyCS was activated by MgCl2 and CaCl2, which inhibit various bacterial CSs. SyCS was not inhibited by ATP and NADH; which are typical feedback inhibitors of other bacterial CSs. SyCS was inhibited by phosphoenolpyruvate and activated by ADP, which has not been reported for CSs from heterotrophic bacteria. Thus, SyCS showed unique characteristics, particularly its sensitivity to effectors.

DOI： 10.1038/s41598-019-42659-z

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Language：English   Publisher：Society for Biotechnology  

DOI： 10.1016/j.jbiosc.2018.07.019

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DOI： 10.1016/j.algal.2018.11.017

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In the oxidative pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) is one of the enzymes that catalyzes reactions generating NADPH. The model cyanobacterium Synechocystis sp. PCC 6803 is widely studied for numerous applications; however, biochemical knowledge of the NADPH production pathway in Synechocystis sp. PCC 6803 is limited. In this study, we conducted biochemical analysis of a 6-phosphogluconate dehydrogenase from Synechocystis sp. PCC 6803 (Sy6PGDH). We found that Sy6PGDH has unconventional characteristics, i.e. the highest kcat value and non-competitive inhibition by NADPH. Additionally, phylogenetic analysis of cyanobacterial 6PGDHs revealed that an amino acid residue at position 42 in Sy6PGDH is highly conserved for each order of cyanobacteria, but Sy6PGDH is phylogenetically unique. In Sy6PGDH, a single amino acid substitution at position 42 from serine to threonine enhanced the affinity for NADP+ and altered the mode of inhibition by NADPH. The amino acid substitution equivalent to Ser42 also altered the affinity for NADP+ and mode of inhibition by NADPH in Arthrospira platensis. These data suggested that an amino acid residue corresponding to position 42 in Sy6PGDH is one of the important residues that possibly determines the function of cyanobacterial 6PGDHs.

DOI： 10.1093/pcp/pcy165

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DOI： 10.1016/j.jbiosc.2018.02.005

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DOI： 10.1093/pcp/pcx162

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DOI： 10.1007/978-981-13-0854-3_7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

DOI： 10.3390/molecules23051051

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Language：English   Publishing type：Research paper (scientific journal)  

Cyanobacteria possess an atypical tricarboxylic acid (TCA) cycle with various bypasses. Previous studies have suggested that a cyclic flow through the TCA cycle is not essential for cyanobacteria under normal growth conditions. The cyanobacterial TCA cycle is, thus, different from that in other bacteria, and the biochemical properties of enzymes in this TCA cycle are less understood. In this study, we reveal the biochemical characteristics of malate dehydrogenase (MDH) from Synechocystis sp. PCC 6803 MDH (SyMDH). The optimal temperature of SyMDH activity was 45-50°C and SyMDH was more thermostable than MDHs from other mesophilic microorganisms. The optimal pH of SyMDH varied with the direction of the reaction: pH 8.0 for the oxidative reaction and pH 6.5 for the reductive reaction. The reductive reaction catalysed by SyMDH was activated by magnesium ions and fumarate, indicating that SyMDH is regulated by a positive feedback mechanism. The Km-value of SyMDH for malate was approximately 210-fold higher than that for oxaloacetate and the Km-value for NAD+ was approximately 19-fold higher than that for NADH. The catalytic efficiency of SyMDH for the reductive reaction, deduced from kcat-values, was also higher than that for the oxidative reaction. These results indicate that SyMDH is more efficient in the reductive reaction in the TCA cycle, and it plays key roles in determining the direction of the TCA cycle in this cyanobacterium.

DOI： 10.3389/fpls.2018.00947

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-017-15341-5

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DOI： 10.1093/pcp/pcx033

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DOI： 10.3389/fmicb.2017.00118

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DOI： 10.1038/srep41080

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本農芸化学会  

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmicb.2016.02050

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DOI： 10.1016/j.algal.2016.06.026

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DOI： 10.1038/srep32354

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer New York  

DOI： 10.1007/978-3-319-25979-6_20

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1186/s12934-016-0416-8

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DOI： 10.1016/j.jbiosc.2015.03.004

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmicb.2015.01064

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmicb.2015.01150

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmicb.2015.00888

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2014.12.020

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1462-2920.12715

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DOI： 10.3389/fmicb.2015.00326

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DOI： 10.1021/bi1501560h

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Language：Japanese   Publisher：The Surface Science Society of Japan  

Biotechnology of cyanobacteria takes much attention because of their ability to utilize light energy and assimilate carbon dioxide. <i>Synechocystis</i> species are unicellular, non-nitrogen fixing cyanobacteria, which are widely used for both basic and applied sciences. Here I introduce our study discovering new way to increase the productivity of hydrogen from <i>Synechocystis</i> cells. SigE, an RNA polymerase sigma factor, is a positive regulator of sugar catabolism and bioplastic production. The <i>sigE</i> overexpression also increases the hydrogen production during dark anaerobic conditions. Phenotypic analysis also reveals that the <i>sigE</i> overexpression alters the photosynthetic activity and cell sizes, indicating the close interaction among metabolism, photosynthesis, cell size maintenance, and bioproduction. These results demonstrate the novel method promoting hydrogen productivity using transcriptional regulator of cyanobacteria.

DOI： 10.1380/jsssj.36.86

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Other Link： https://jlc.jst.go.jp/DN/JLC/20005429583?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Hindawi Limited  

DOI： 10.1155/2014/921976

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DOI： 10.1111/1462-2920.12554

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DOI： 10.1104/pp.113.232025

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DOI： 10.1111/1462-2920.12170

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DOI： 10.1016/j.febslet.2013.12.008

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DOI： 10.1016/j.febslet.2013.12.008

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DOI： 10.1093/dnares/dst028

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DOI： 10.1111/tpj.12310

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DOI： 10.1074/jbc.M111.231183

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DOI： 10.1093/pcp/pcq204

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DOI： 10.1073/pnas.0810040106

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Language：English  

DOI： 10.1111/j.1399-3054.2008.01078.x

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DOI： 10.1093/pcp/pcm072

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Language：English  

DOI： 10.1039/b616219n

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/dnares/ds1010

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DOI： 10.1074/jbc.M507489200

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DOI： 10.1074/jbc.M505043200

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Applicant：国立研究開発法人理化学研究所

Application no：JP2019018861  Date applied：2019.5

Publication no：WO2020-054126  Date published：2020.3

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Applicant：学校法人明治大学, 株式会社ユーグレナ

Application no：特願2018-201244  Date applied：2018.10

Announcement no：特開2019-110894  Date announced：2019.7

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Applicant：学校法人明治大学, 株式会社ユーグレナ

Application no：特願2018-201244  Date applied：2018.10

Announcement no：特開2019-110894  Date announced：2019.7

Patent/Registration no：特許第7274851号  Date registered：2023.5 

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Applicant：学校法人明治大学, 株式会社ユーグレナ

Application no：特願2017-105892  Date applied：2017.5

Announcement no：特開2018-198580  Date announced：2018.12

Patent/Registration no：特許第6925169号  Date registered：2021.8 

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Applicant：国立研究開発法人理化学研究所, 株式会社ユーグレナ

Application no：特願2017-517913  Date applied：2016.5

Patent/Registration no：特許第6745513号  Date registered：2020.8 

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Applicant：国立研究開発法人理化学研究所

Application no：特願2016-574833  Date applied：2016.2

Patent/Registration no：特許第6778870号  Date registered：2020.10 

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Applicant：国立研究開発法人理化学研究所

Application no：JP2016053972  Date applied：2016.2

Announcement no：WO2016-129636  Date announced：2016.8

Patent/Registration no：特許第6778870号  Date registered：2020.10 

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Applicant：学校法人明治大学

Application no：特願2016-005334  Date applied：2016.1

Announcement no：特開2017-123816  Date announced：2017.7

Patent/Registration no：特許第6681065号  Date registered：2020.3 

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Applicant：学校法人明治大学

Application no：特願2015-200116  Date applied：2015.10

Announcement no：特開2017-070252  Date announced：2017.4

Patent/Registration no：特許第6688497号  Date registered：2020.4 

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Applicant：国立研究開発法人理化学研究所

Application no：特願2015-560001  Date applied：2015.1

Patent/Registration no：特許第6249456号  Date issued：2017.12

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Applicant：国立研究開発法人理化学研究所

Application no：特願2015-505453  Date applied：2014.3

Patent/Registration no：特許第5946080号  Date issued：2016.6

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