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.
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.
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.
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