Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803.

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.

Simultaneous increases in the levels of compatible solutes by cost-effective cultivation of Synechocystis sp. PCC 6803.

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.

Cluster-Level Relationships of Genes Involved in Carbon Metabolism in Synechocystis sp. PCC 6803: Development of a Novel Succinate-Producing Strain.

We quantified the transcript levels of 44 genes related to sugar catabolism in strains with altered primary carbon metabolism and discovered a consistent expression pattern among succinate-producing mutants. To identify factors that determine the expression pattern, we calculated Pearson's correlation coefficients, using the transcript data. Correlation analysis revealed positive and negative correlations among genes encoding sugar catabolic enzymes. On the basis of this analysis, we found that the mutant overexpressing both rre37 (encoding an OmpR-type response regulator) and sigE (encoding an RNA polymerase sigma factor) produced increased levels of succinate under dark, anaerobic conditions, with a maximum productivity of 420 mg l-1.

Corrigendum: Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle.

[This corrects the article DOI: 10.3389/fpls.2018.00947.].

Fumarase From Cyanidioschyzon merolae Stably Shows High Catalytic Activity for Fumarate Hydration Under High Temperature Conditions.

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.

Genetic manipulation to overexpress rpaA altered photosynthetic electron transport in Synechocystis sp. PCC 6803.

Cyanobacteria are a group of prokaryotic organisms that perform oxygenic photosynthesis using a similar photosynthetic apparatus as is used in higher plants and eukaryotic algae. Cyanobacteria are also known to have a circadian rhythm. Here, we evaluated the effects on photosynthesis caused by the genetic manipulation of RpaA, which is a response regulator of a two-component regulatory system responsible for the signal output from circadian clocks. Using the unicellular cyanobacterium Synechocystis sp. PCC 6803, photosynthetic activities and transcript levels of photosystem I and photosystems II in the rpaA-overexpressing strain were measured, and it was found that the parameters, such as Fv/Fm, Fv'/Fm', qP, and ϕII, obtained from chlorophyll fluorescence analysis were decreased by rpaA overexpression. These results suggest that rpaA overexpression modified photosynthetic electron transport under normal light conditions. Thus, we demonstrated that RpaA regulates photosynthesis in cyanobacteria and can be a potential target of photosynthetic engineering in this cyanobacterium.

Purification and Characterisation of Malate Dehydrogenase From Synechocystis sp. PCC 6803: Biochemical Barrier of the Oxidative Tricarboxylic Acid Cycle.

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.

Gut Microbiota-Derived Short Chain Fatty Acids Induce Circadian Clock Entrainment in Mouse Peripheral Tissue.

Microbiota-derived short-chain fatty acids (SCFAs) and organic acids produced by the fermentation of non-digestible fibre can communicate from the microbiome to host tissues and modulate homeostasis in mammals. The microbiome has circadian rhythmicity and helps the host circadian clock function. We investigated the effect of SCFA or fibre-containing diets on circadian clock phase adjustment in mouse peripheral tissues (liver, kidney, and submandibular gland). Initially, caecal SCFA concentrations, particularly acetate and butyrate, induced significant day-night differences at high concentrations during the active period, which were correlated with lower caecal pH. By monitoring luciferase activity correlated with the clock gene Period2 in vivo, we found that oral administration of mixed SCFA (acetate, butyrate, and propionate) and an organic acid (lactate), or single administration of each SCFA or lactate for three days, caused phase changes in the peripheral clocks with stimulation timing dependency. However, this effect was not detected in cultured fibroblasts or cultured liver slices with SCFA applied to the culture medium, suggesting SCFA-induced indirect modulation of circadian clocks in vivo. Finally, cellobiose-containing diets facilitated SCFA production and refeeding-induced peripheral clock entrainment. SCFA oral gavage and prebiotic supplementation can facilitate peripheral clock adjustment, suggesting prebiotics as novel therapeutic candidates for misalignment.

Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC.

Succinate and lactate are commodity chemicals used for producing bioplastics. Recently, it was found that such organic acids are excreted from cells of the unicellular cyanobacterium Synechocystis sp. PCC 6803 under dark, anaerobic conditions. To conduct the dark, anaerobic incubation, cells were concentrated within a vial that was then sealed with a butyl rubber cap, following which N2 gas was introduced into the vial. The organic acids produced were quantified by high-performance liquid chromatography via post-labeling with bromothymol blue as a pH indicator. After separation by ion-exclusion chromatography, the organic acids were identified by comparing their retention time with that of standard solutions. These procedures allow researchers to quantify the organic acids produced by microorganisms, contributing to knowledge about the biology and biotechnology of cyanobacteria.

Polyporus and Bupleuri radix effectively alter peripheral circadian clock phase acutely in male mice.

In mammals, daily physiological events are precisely regulated by an internal circadian clock system. An important function of this system is to readjust the phase of the clock daily. In Japan, traditional herb medicines, so-called crude drugs (Shoyaku), are widely used for many diseases, and some are reported to affect circadian clock impairment, suggesting that some of them might have an ability to modify clock gene expression rhythms. Therefore, from selected 40 crude drugs, finding candidates that control the circadian clock phases was the first purpose of this study. As there are several crude drugs used for liver- and/or kidney-related diseases, the second aim of the present study was to find some crude drugs affecting liver/kidney circadian clock in vivo. To assess phase changes in the daily circadian rhythm, bioluminescence from the core clock gene product Period 2 was continuously monitored in mouse embryonic fibroblasts in vitro and in some peripheral tissues (kidney, liver, and submandibular gland) of PERIOD2::LUCIFERASE knock-in mice in vivo. In our screening, Polyporus and Bupleuri radix were found to be good candidates to effectively manipulate the peripheral circadian clock phase acutely, with stimulation time-of-day dependency in vitro as well as in vivo. Interestingly, Polyporus and Bupleuri radix are traditional herb medicines use for treating edema and promoting diuresis, and for chronic hepatitis, respectively. These crude drugs may be therefore good modulators of the circadian peripheral clocks including liver and kidney, and circadian clock genes become new molecular targets for these crude drugs.