Process engineering of pH tolerant Ustilago cynodontis for efficient itaconic acid production

Abstract

BackgroundUstilago cynodontis ranks among the relatively unknown itaconate production organisms. In comparison to the well-known and established organisms like Aspergillus terreus and Ustilago maydis, genetic engineering and first optimizations for itaconate production were only recently developed for U. cynodontis, enabling metabolic and morphological engineering of this acid-tolerant organism for efficient itaconate production. These engineered strains were so far mostly characterized in small scale shaken cultures.ResultsIn pH-controlled fed-batch experiments an optimum pH of 3.6 could be determined for itaconate production in the morphology-engineered U. cynodontis Δfuz7. With U. cynodontis ∆fuz7r ∆cyp3r PetefmttA Pria1ria1, optimized for itaconate production through the deletion of an itaconate oxidase and overexpression of rate-limiting production steps, titers up to 82.9 ± 0.8 g L−1 were reached in a high-density pulsed fed-batch fermentation at this pH. The use of a constant glucose feed controlled by in-line glucose analysis increased the yield in the production phase to 0.61 gITA gGLC−1, which is 84% of the maximum theoretical pathway yield. Productivity could be improved to a maximum of 1.44 g L−1 h−1 and cell recycling was achieved by repeated-batch application.ConclusionsHere, we characterize engineered U. cynodontis strains in controlled bioreactors and optimize the fermentation process for itaconate production. The results obtained are discussed in a biotechnological context and show the great potential of U. cynodontis as an itaconate producing host.

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 [24][25][26][27]. This, along with advances in bioprocess design enabled by the yeast morphology [27][28][29], has considerably enhanced the yield, titer, and rate of itaconic acid production by Ustilago.…”

Consolidated bioprocessing of cellulose to itaconic acid by a co-culture of Trichoderma reesei and Ustilago maydis.

“…maydis, the residual NH 4 + -concentration present in the cellulase-containing supernatants had to be monitored (50). 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 (26,28,29). This resulted in a nal cellulase titer of 2.2 FPU/mL for the cultivations containing T. reesei supernatant and 0.6 FPU/mL for the cultivations containing P. verruculosum supernatant.…”

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).…”