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

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.

AnUstilago maydischassis for itaconic acid production without by‐products

“…As last modification, ria1, encoding the itaconate cluster regulator, was overexpressed by a promoter exchange to improve itaconate and decrease malate production (Geiser et al, 2016b). This was achieved by the inclusion of the P etef promoter into the repair template yielding a marker-free insertion (Hosseinpour Tehrani et al, 2019a). For the generation of Δcyp3, ΔMEL, ΔUA and ΔP ria1 ::P etef , the CRISPR/Cas9 system was used, while Δdgat was achieved using the FRT/FLP-mediated approach.…”

A Review of the Biotechnological Production of Methacrylic Acid

“…Itaconic acid is also produced from citrate through the intermediate cis-aconitate which is decarboxylated to itaconate as illustrated in Figure 1M. Titers in the range of 120-220 g/L have been reported with itaconate yields ranging from 0.45 to as high as 0.58 g itaconic acid/ g glucose and maximal production rates from 0.45 to 1g/L-h (Hevekerl et al, 2014;Huang et al, 2014;Krull et al, 2017a;Tehrani et al, 2019). In addition, the chemical decarboxylation of itaconic acid to MA has been demonstrated via several different catalytic routes, mostly reliant on metal catalysts.…”

“…The route to MA through itaconic acid may well be the most mature, with previous scale up and commercial production but key improvements to reduce costs would include improving the yield of fermentation, as well as increasing the volumetric rate of production by at least 2 fold (Bafana and Pandey, 2018). Recent efforts have been aimed at engineering organisms beyond A. terreus including U. maydis, Y. lipolytica, and E. coli (Krull et al, 2017b;Tehrani et al, 2019;Zhao et al, 2019).…”

Consolidated Bioprocessing of Cellulose to Itaconic Acid by a Co-Culture of Trichoderma Reesei and Ustilago Maydis.

“…Recently, itaconic acid production with U. maydis and other Ustilaginaceae such as U. cynodontis has been improved considerably by genetic engineering (16)(17)(18)(19). This, along with advances in bioprocess design enabled by the yeast morphology (19)(20)(21), has considerably enhanced the yield, titer, and rate of itaconic acid production by Ustilago.…”

“…Since nitrogen limitation is a prerequisite for itaconic acid production with U. maydis, the residual NH 4 + -concentration present in the cellulase-containing supernatants had to be monitored (42). The supernatants were diluted accordingly to reach a nal NH 4 + -concentration equivalent to the 0.8 g/L NH 4 Cl, typically present in itaconic acid production medium for U. maydis (18,20,21).…”