Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production.

The Ustilaginaceae family of smut fungi, especially Ustilago maydis, gained biotechnological interest over the last years, amongst others due to its ability to naturally produce the versatile bio-based building block itaconate. Along with itaconate, U. maydis also produces 2-hydroxyparaconate. The latter was proposed to be derived from itaconate, but the underlying biochemistry and associated genes were thus far unknown. Here, we confirm that 2-hydroxyparaconate is a secondary metabolite of U. maydis and propose an extension of U. maydis' itaconate pathway from itaconate to 2-hydroxyparaconate. This conversion is catalyzed by the P450 monooxygenase Cyp3, encoded by cyp3, a gene, which is adjacent to the itaconate gene cluster of U. maydis. By deletion of cyp3 and simultaneous overexpression of the gene cluster regulator ria1, it was possible to generate an itaconate hyper producer strain, which produced up to 4.5-fold more itaconate in comparison to the wildtype without the by-product 2-hydroxyparaconate. By adjusting culture conditions in controlled pulsed fed-batch fermentations, a product to substrate yield of 67% of the theoretical maximum was achieved. In all, the titer, rate and yield of itaconate produced by U. maydis was considerably increased, thus contributing to the industrial application of this unicellular fungus for the biotechnological production of this valuable biomass derived chemical.

Enhanced malic acid production from glycerol with high-cell density Ustilago trichophora TZ1 cultivations.

BACKGROUND: In order to establish a cost-efficient biodiesel biorefinery, valorization of its main by-product, crude glycerol, is imperative. Recently, Ustilago trichophora TZ1 was found to efficiently produce malic acid from glycerol. By adaptive laboratory evolution and medium optimization, titer and rate could be improved significantly. RESULTS: Here we report on the investigation of this strain in fed-batch bioreactors. With pH controlled at 6.5 (automatic NaOH addition), a titer of 142 ± 1 g L(-1) produced at an overall rate of 0.54 ± 0.00 g L(-1) h(-1) was reached by optimizing the initial concentrations of ammonium and glycerol. Combining the potential of bioreactors and CaCO3 as buffer system, we were able to increase the overall production rate to 0.74 ± 0.06 g L(-1) h(-1) with a maximum production rate of 1.94 ± 0.32 g L(-1) reaching a titer of 195 ± 15 g L(-1). The initial purification strategy resulted in 90 % pure calcium malate as solid component. Notably, the fermentation is not influenced by an increased temperature of up to 37 °C, which reduces the energy required for cooling. However, direct acid production is not favored as at a lowered pH value of pH 4.5 the malic acid titer decreased to only 9 ± 1 g L(-1). When using crude glycerol as substrate, only the product to substrate yield is decreased. The results are discussed in the context of valorizing glycerol with Ustilaginaceae. CONCLUSIONS: Combining these results reveals the potential of U. trichophora TZ1 to become an industrially applicable production host for malic acid from biodiesel-derived glycerol, thus making the overall biodiesel production process economically and ecologically more feasible.

Efficient malic acid production from glycerol with Ustilago trichophora TZ1.

BACKGROUND: The large surplus of crude glycerol, as main low-value waste stream in biodiesel production, has led to the investigation of new possibilities for the production of value-added chemicals from this feedstock. New and efficient (bio-) catalysts are needed that are able to convert glycerol to versatile chemical building blocks. This would contribute to further develop away from a mainly petroleum based, to a sustainable, bio-based industry. One promising group of discussed building block chemicals are dicarbonic acids. RESULTS: Here, we report the efficient synthesis of malate from glycerol using Ustilago trichophora RK089, which was identified in a screening of 74 Ustilaginaceae. For economically feasible production that can compete with existing processes, a high productivity is required. By adaptive laboratory evolution, the growth and production rate were increased by 2.5- and 6.6-fold, respectively. Further medium optimization increased the final titer, yield, and overall production rate to 196 g L(-1), 0.82 gmal ggly (-1), and 0.39 g L(-1) h(-1), respectively. CONCLUSIONS: This titer is the highest reported for microbial malate production, making U. trichophora TZ1 a promising microbial production host for malate from crude glycerol, especially since it is not genetically engineered. Since this production process starts from an industrial waste stream as substrate and yields an interesting platform chemical, which can be used to replace petro-chemicals, it greatly contributes to a sustainable bio-economy.