Itaconic Acid and Beyond: New Insights into the Itaconic Acid Production Pathway of Ustilago Maydis

Itaconic acid is a bio-based platform chemical for the production of pharmaceuticals, adhesives and polymers. Ustilago is a promising fungal host for the production of itaconic acid [1]. Contrary to established filamentous itaconate producers, Ustilago grows unicellularly. This has distinct process advantages relating to morphology control, viscosity and aeration [2]. However, the itaconate production yield, titer and rate of Ustilago are thusfar below those of the commercial production hosts. In order to optimize itaconate production in Ustilago by metabolic engineering, detailed biochemical knowledge was needed.

We recently identified an itaconate gene cluster in Ustilago. The function of the cluster genes and their encoded proteins was characterized by knockout and overexpression. The activity of two essential catalytic proteins was determined in vivo by permeabilized cell assays and in vitro by E. coli expression and purification. In contrast to the known itaconate biosynthesis pathway of Aspergillus terreus, Ustilago’s itaconate production proceeds first via an isomerization from cis- to trans-aconitate, followed by decarboxylation of the trans-aconitate [3].

Two additional genes in the cluster, cyp3 (cytochrome P450 monooxygenase) and rdo1 (ring-cleaving dioxygenase) are not directly involved in itaconate production. Instead, they appear to have a role in the further conversion of itaconate, likely into the more oxidized products 2-hydroxyparaconate and itatartarate. Although these products have been identified previously [2,4], the associated genes and pathway was until now unclear. We here present the application of this new biochemical knowledge to enhance the overall product titer and yield, but also to precisely control product specificity. The results are discussed in the context of the process advantages of smut fungi as novel hosts for industrial biotechnology.

References

[1] N. Maassen, M. Panakova, N. Wierckx, E. Geiser, M. Zimmermann, M. Bölker, U. Klinner, L.M. Blank. 2014. Influence of carbon and nitrogen concentration on itaconic acid production by the smut fungus Ustilago maydis. Eng. Life Sci. 14:129-134.

[2] E. Geiser, V. Wiebach, N. Wierckx, L.M. Blank. 2014. Prospecting the biodiversity of the fungal family Ustilaginaceae for the production of value-added chemicals. Fung. Biol. Biotechnol. 1:2

[3] E. Geiser, S.K. Przybilla, A. Friedrich, W. Buckel, N. Wierckx, L.M. Blank, M. Bölker. 2016. Ustilago maydis produces itaconic acid via the unusual intermediate trans-aconitate. Microb. Biotechnol. 9: 116-126

[4] Guevarra, E.D. and Tabuchi, T. (1990) Accumulation of itaconic, 2-hydroxyparaconic, itatartaric and malic acids by strain of the genus Ustilago. Agric. Biol. Chem. 54:2353