Integrated strain- and process design enable production of 220 g L-1 itaconic acid with Ustilago maydis

Hamed Hosseinpour Tehrani; Johanna Becker; Isabel Bator; Katharina Saur; Svenja Meyer; Ana Catarina Rodrigues Lóia; Lars M Blank; Nick Wierckx

doi:10.1186/s13068-019-1605-6

Integrated strain- and process design enable production of 220 g L^-1 itaconic acid with Ustilago maydis

Biotechnol Biofuels. 2019 Nov 6:12:263. doi: 10.1186/s13068-019-1605-6. eCollection 2019.

Authors

Hamed Hosseinpour Tehrani¹, Johanna Becker¹, Isabel Bator¹, Katharina Saur¹, Svenja Meyer¹, Ana Catarina Rodrigues Lóia¹, Lars M Blank¹, Nick Wierckx^{1

2}

Affiliations

¹ 1iAMB-Institute of Applied Microbiology, ABBt-Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany.
² 2Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich, Wilhelm-Johnen-Str., 52425 Jülich, Germany.

Abstract

Background: Itaconic acid is an unsaturated, dicarboxylic acid which finds a wide range of applications in the polymer industry and as a building block for fuels, solvents and pharmaceuticals. Currently, Aspergillus terreus is used for industrial production, with titers above 100 g L^-1 depending on the conditions. Besides A. terreus, Ustilago maydis is also a promising itaconic acid production host due to its yeast-like morphology. Recent strain engineering efforts significantly increased the yield, titer and rate of production.

Results: In this study, itaconate production by U. maydis was further increased by integrated strain- and process engineering. Next-generation itaconate hyper-producing strains were generated using CRISPR/Cas9 and FLP/FRT genome editing tools for gene deletion, promoter replacement, and overexpression of genes. The handling and morphology of this engineered strain were improved by deletion of fuz7, which is part of a regulatory cascade that governs morphology and pathogenicity. These strain modifications enabled the development of an efficient fermentation process with in situ product crystallization with CaCO₃. This integrated approach resulted in a maximum itaconate titer of 220 g L^-1, with a total acid titer of 248 g L^-1, which is a significant improvement compared to best published itaconate titers reached with U. maydis and with A. terreus.

Conclusion: In this study, itaconic acid production could be enhanced significantly by morphological- and metabolic engineering in combination with process development, yielding the highest titer reported with any microorganism.

Keywords: Biochemical engineering; In situ precipitation; Itaconic acid; Metabolic engineering; Morphological engineering; Ustilago maydis.