Abstract
BackgroundItaconic 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.ResultsIn 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.ConclusionIn 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.
Highlights
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
The overexpression of ria1 was not achieved by the in trans insertion of an expression cassette at the cbx locus, but rather by a direct in cis replacement of the native native promoter of ria1 (Pria1) promoter by the strong and constitutive strong and constitutive promoter (Petef) promoter
In this study, the combination of metabolic and morphological engineering together with in situ crystallization of itaconate yielded a titer of 220 g L−1 itaconate, which corresponds to 284 g L−1 calcium itaconate
Summary
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. Hosseinpour Tehrani et al Biotechnol Biofuels (2019) 12:263 production by mammalian macrophages is reported, where it plays a key role in the human immune response [11, 57, 69], with possible applications as therapeutic agent for autoimmune diseases [2] In spite of this wide variety of potential applications, the market size of itaconic acid in 2011 was relatively small, with 41,400 tons and a market value of $74.5 million [71]. This is caused by the relatively high price of approximately two dollars per kg and the availability of cheaper petro-based alternatives such as acrylic acid. Morphological control influences the manufacturing process tremendously, leading to increased operational costs and failed batches
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