Journal
BIOTECHNOLOGY FOR BIOFUELS
Volume 12, Issue 1, Pages -Publisher
BMC
DOI: 10.1186/s13068-019-1605-6
Keywords
Ustilago maydis; Itaconic acid; Metabolic engineering; Morphological engineering; Biochemical engineering; In situ precipitation
Funding
- German Federal Ministry of Food and Agriculture (BMEL), through the Specialist agency renewable raw materials e.V. (FNR) as part of the ERA-IB project TTRAFFIC [FKZ 22030515]
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [236, 2186]
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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.
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