Metabolic Engineering von Corynebacterium glutamicum für die Produktion einer Dicarbonsäure

Due to its chemical and physical properties and its broad range of applications, itaconic acid was classified as a promising building block molecule for the chemical industry that can be produced from renewable feedstocks like glucose. Biotechnologically produced itaconate might be able to replace chemicals derived from petroleum and thus can contribute to sustainability and a reduction of environmental pollution. In this work, metabolic engineering was used to design and develop itaconate-producing Corynebacterium glutamicum strains. The following results were obtained: 1. Itaconate is formed from cis-aconitate, an intermediate of the aconitase reaction converting citrate to isocitrate, by cis-aconitate decarboxylase (CAD). As C. glutamicum does not contain this enzyme, the cad gene from Aspergillus terreus was heterologously expressed in C. glutamicum wild type. The resulting strain secreted 1.4 mM of itaconate into the medium, confirming the functionality of CAD. However, the specific CAD activity in crude extracts was much lower than in A. terreus. Interestingly, itaconate was produced by C. glutamicum only in the stationary growth phase. 2. CAD activity could be increased by fusing the enzyme to the maltose binding protein MalE of Escherichia coli lacking its signal peptide. The strain containing the MalE-Cad fusion protein produced 3.6 mM of itaconate in the stationary phase. 3. To improve the glucose supply for itaconate production in the stationary phase, cells were grown under nitrogen-limitation. This led to reduced biomass formation and the presence of residual glucose after growth was increased. With this strategy C. glutamicum accumulated 30 mM of itaconate in the medium. The enhancement was attributed to an increased CAD activity in the crude extract and improved sugar availability. 4. To optimize the carbon flux towards itaconic acid production, isocitrate dehydrogenase activity (ICD) was reduced by a start codon exchange. In this way, the itaconate titer in the medium was increased to 60 mM. 5. In the last step, itaconate production was scaled up successfully from shake flasks with 60 ml medium to a bioreactor system with 600 ml medium. This process exposed the importance of dissolved oxygen for itaconate production by C. glutamicum.