Biochemical elucidation of citrate accumulation in Synechocystis sp. PCC 6803 via kinetic analysis of aconitase

Abstract

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.

Sustainable citric acid production from CO2 in an engineered cyanobacterium

“…As shown in Figures 3, 6, a simple overexpression of citrate synthase, even with a strong constitutive promoter ("push"), is not sufficient to generate enough metabolic overflow to citric acid and enable either its accumulation in the cell or excretion to the growth medium. The cyanobacterial aconitase, as recently shown for the Syn6803 enzyme, has a K m for citric acid of 1.1 mM (Nishii et al, 2021). While 10-fold lower than the corresponding E. coli counterpart, the flux through aconitase was seemingly enough to accommodate an increase in the flux through the citrate synthase node, even when different citrate synthases were overexpressed, resulting in no significant citric acid accumulation.…”

Proteomics analysis reveals the inactivation mechanisms of Alicyclobacillus acidoterrestris spores by atmospheric cold plasma

“…In C/C2, the enzyme showed significant up‐regulation, moreover, haem, which plays a role in transferring electrons in the electron transport chain, is catalytically generated by uroporphyrinogen decarboxylase (T0DN60), which is up‐regulated in C/Ct2 with a FC of 2.44, indicating that energy metabolism within AAT spores in the treatment group received limited inhibition (Simão‐Gurge et al ., 2019). The citric acid (TCA) cycle is the largest source of energy in living organisms; Aconitase hydratase (A0A512CMM3), which catalyses the isomerisation of citrate to isocitrate in the TCA cycle (Nishii et al ., 2021), was up‐regulated in C compared to Ct1, in addition, fumarylacetoacetate (FAA) hydrolase (T0BQU5) as well as malate dehydrogenase (T0BTR2), both process enzymes of the TCA cycle, were up‐regulated in C compared to Ct2. Thus, energy metabolism within spores is severely inhibited as a response to environmental stress.…”

“…Ó 2023 Institute of Food, Science and Technology (IFSTTF). (Nishii et al, 2021), was up-regulated in C compared to Ct1, in addition, fumarylacetoacetate (FAA) hydrolase (T0BQU5) as well as malate dehydrogenase (T0BTR2), both process enzymes of the TCA cycle, were up-regulated in C compared to Ct2. Thus, energy metabolism within spores is severely inhibited as a response to environmental stress.…”

An unexpected role for leucyl aminopeptidase in UV tolerance revealed by a genome-wide fitness assessment in a model cyanobacterium

“…P68767), called lap in Synechocystis sp. PCC 6803 ( 16 ), which encodes a putative leucyl aminopeptidase. We assessed the phenotype of a knockout mutant of S. elongatus Synpcc7942_1190 , hereafter called lap , and performed kinetic assays of the LAP protein.…”