Citric acid is one of the most important organic acids produced by fermentation with the filamentous fungus Aspergillus niger using glucose as substrate. This organic acid is extensively used in food, cosmetic and pharmaceutical industries with a production capacity of nearly 2 million tons per year (Steiger et al., 2017). Citric acid is produced in the tricarboxylic acid cycle (TCA) from oxaloacetate and acetyl-CoA. In glycolysis, 1 mol glucose is oxidized and converted into 2 mol of pyruvate. 1 mol of pyruvate is carboxylated to oxaloacetate, the other is decarboxylated to acetyl-CoA, resulting in the net reaction:

Glucose + 3 NAD+ + H2O ⇌ Citrate + 3 NADH

While this pathway leads to a theoretically balanced carbon yield it is not redox balanced, and re-oxidizing the NADH leads to high oxygen consumption and heat release (Karaffa & Kubicek, 2003). Mixed-substrate conversion allows to incorporate CO2, a cheap carbon source, into products (e.g. organic acids) with higher oxidation states than the co-substrate (e.g. glucose). This is a promising strategy to fix CO2 in an industrial process and increase the total carbon yield of the process without requiring oxygen as an electron acceptor, which reduces the need for extensive cooling (Steiger et al., 2017).

Therefore, the aim of this PhD thesis is to increase the carbon efficiency of citric acid production by developing a synthetic pathway avoids decarboxylation leading to a net CO2 assimilation during the mixed-substrate production of citric acid. The pathway will be incorporated in the yeast Komagataella phaffii (Pichia pastoris) to create an orthogonal test system. Therefore, the citric acid transporter genes of Yarrowia lipolytica (Erian et al., 2020) or Aspergillus niger (Steiger et al. 2019) will be expressed in K. phaffii, and the orthogonal pathway realized by expressing the respective genes under control of methanol regulated promoters. After evaluation the best pathway variants will be transferred into A. niger.


Erian, A. M., Egermeier, M., Rassinger, A., Marx, H., & Sauer, M. (2020). Identification of the citrate exporter Cex1 of Yarrowia lipolytica. FEMS Yeast Research, 20(7), 1–10.

Karaffa, L., & Kubicek, C. (2003). Aspergillus niger citric acid accumulation: do we understand this well working black box? Appl Microbiol Biotechnol, 61, 189–196.

Rußmayer, H., Buchetics, M., Gruber, C., Valli, M., Grillitsch, K., Modarres, G., Guerrasio, R., Klavins, K., Neubauer, S., Drexler, H., Steiger, M., Troyer, C., Chalabi, A. Al, Krebiehl, G., Sonntag, D., Zellnig, G., Daum, G., Graf, A. B., Altmann, F., … Gasser, B. (2015). Systems-level organization of yeast methylotrophic lifestyle.

Steiger, M. G., Mattanovich, D., & Sauer, M. (2017). Microbial organic acid production as carbon dioxide sink. FEMS Microbiology Letters, October, 1–4.

Steiger, M. G., Rassinger, A., Mattanovich, D., & Sauer, M. (2019). Engineering of the citrate exporter protein enables high citric acid production in Aspergillus niger. Metabolic Engineering, 52(November 2018), 224–231.