Biosynthesis of itaconic acid. Glucose is taken up from the medium and converted into pyruvate via glycolysis. Pentose sugars are converted to pyruvate via pentose phosphate pathway (PPP). Box shows the reaction stoichiometry and resulting respiratory quotients (RQ) using different pathways: Pathway a: To reach the maximum theoretical yield of 1 mol/mol glucose, itaconic synthesis must involve anaplerotic CO2 fixation: for each mol of glucose, 1 mol of the produced pyruvate is transported to the mitochondrion and 1 mol of pyruvate is converted to malate in the cytosol (1a). In the mitochondrion, pyruvate is converted to acetyl-CoA (1b) and further to citrate by citrate synthase (2) using oxaloacetate. Citrate is then converted into cis-aconitate by aconitase (3). Via a mitochondrial tricarboxylate-malate shuttle (4), cis-aconitate is exported to the cytosol in exchange for malate, which replenishes the oxaloacetate used for citrate synthesis. In the cytosol cis-aconitate is then directly decarboxylated to itaconic acid by cis-aconitate decarboxylase (5) in the case of A. terreus or first isomerized into transaconitate before being decarboxylated in the case of U. maydis. Finally, itaconic acid is exported outside the cell (6). The respiratory quotient for this pathway would be 0.67. Pathway b: If itaconic acid is produced without anaplerotic CO2 fixation, the pathway has to be exclusively fed from acetyl-CoA (1a). In this case, the spent C4 acids such as oxaloacetate also have to be replenished by reactions relying on acetyl-CoA. Hence, 1/3 of the carbon is lost into CO2 at pyruvate decarboxylase step, which is why a minimum of 1.5 mol of glucose will then be needed for producing 1 mol of itaconic acid, resulting in a maximum yield of 0.67 mol/mol glucose. The respiratory quotient for this pathway would be 0.89. Pathway c: The RQ for respiration biomass formation can be considered to be 1. Depending on the share of substrate used for respiration or biomass formation, the itaconic acid yield can be anywhere between 0 and 1 mol/mol glucose while the corresponding respiratory quotient would be between 1 and 0.67, respectively. However, when CaCO3 is used in the medium, the itaconic acid will react to calcium itaconate once exported outside the cell, which releases an additional mol of CO2 (7). This leads to an apparent RQ between 1 and 1.33 for pathways a–c, depending on the itaconic acid production yield. The apparent quotient when using CaCO3 is shown in blue letters.