| Popis: |
The rapidly growing need for energy and fuel combined with an increased awareness of the detrimental influence of fossil resources has evoked the investigation of renewable alternatives, such as biodiesel. The production process of biodiesel, however, results in a huge waste stream of crude glycerol, reducing the economic and ecological advantage.The overall aim of this thesis was to establish a production process for organic acids from biodiesel derived glycerol with Ustilaginaceae. The biodiversity within this family is well known and in a screening of 126 Ustilaginaceae, two promising candidates for organic acid production were found - Ustilago trichophora for malic acid production and U. vetiveriae for itaconic acid production. Glycerol uptake and growth rate of both strains were improved by adaptive laboratory evolution. Selection of the best growing single colony for each strain and medium and process optimization drastically improved the production values.The itaconic acid titer of U. vetiveriae TZ1 was increased to 35 g L 1 produced at a production rate of 0.09 g L 1 h 1. Simultaneously about 60 g L 1 malic acid were formed. In first metabolic engineering approaches overexpressing the mitochondrial transporter Mtt1 and the regulator of the itaconic acid gene cluster Ria1, both from U. maydis, the production values could be shifted in favor of itaconic acid increasing it by 1.5 and 2.0 fold, respectively. Simultaneously, the malic acid titer was reduced by 96 % and 61 %, respectively.For U. trichophora TZ1 the malic acid titer was improved to nearly 200 g L 1 produced within 264 h reaching a maximal production rate of 1.53 g L 1 h 1. Since the knowledge on this obscure U. trichophora was scarce, potential native target genes for metabolic engineering were identified after de novo genome sequencing. To enable the improvement of malic acid production with U. trichophora TZ1 by metabolic engineering, existing tools, such as antibiotic markers and promoters, were investigated and adapted to be suitable for creation of overexpression mutants. Using these tools, overexpression mutants for two different malate dehydrogenases (Mdh1 and Mdh2), pyruvate-carboxylase (Pyc) and two different malic acid transporters (Ssu1 and Ssu2) were generated. While overexpression of Pyc did not improve malic acid production, transformants overexpressing Mdh1 and Mdh2 and malic acid transporters Ssu1 and Ssu2 showed an up to 38 % increased malic acid production rate and an up to 54 % increased yield in shake flasks compared to U. trichophora TZ1. In bioreactor cultivations with the mutant overexpressing Ssu2, an increased production rate could not be observed. Due to a drastically lowered optical density, however, this strain had a 29 % higher specific production rate. Additionally, the product yield was improved by 1.4 fold.These results clearly strengthen the applicability of Ustilaginaceae as industrially valuable production organisms. By this valorization of biodiesel derived crude glycerol, the overall biodiesel bio refinery concept is improved on an economic as well as ecological level. |
