Ustilago maydis Metabolic Characterization and Growth Quantification with a Genome-Scale Metabolic Model.

Ustilago maydis is an important plant pathogen that causes corn smut disease and serves as an effective biotechnological production host. The lack of a comprehensive metabolic overview hinders a full understanding of the organism's environmental adaptation and a full use of its metabolic potential. Here, we report the first genome-scale metabolic model (GSMM) of Ustilago maydis (iUma22) for the simulation of metabolic activities. iUma22 was reconstructed from sequencing and annotation using PathwayTools, and the biomass equation was derived from literature values and from the codon composition. The final model contains over 25% annotated genes (6909) in the sequenced genome. Substrate utilization was corrected by BIOLOG phenotype arrays, and exponential batch cultivations were used to test growth predictions. The growth data revealed a decrease in glucose uptake rate with rising glucose concentration. A pangenome of four different U. maydis strains highlighted missing metabolic pathways in iUma22. The new model allows for studies of metabolic adaptations to different environmental niches as well as for biotechnological applications.

Online in vivo monitoring of cytosolic NAD redox dynamics in Ustilago maydis.

Maintenance of metabolic redox homeostasis is essential to all life and is a key factor in many biotechnological processes. Changes in the redox state of NAD affect metabolic fluxes, mediate regulation and signal transduction, and thus determine growth and productivity. Here we establish an in vivo monitoring system for the dynamics of the cytosolic NADH/NAD+ ratio in the basidiomycete Ustilago maydis using the ratiometric fluorescent sensor protein Peredox-mCherry. Metabolic redox dynamics were determined in the cytosol of living cells with high time resolution under biotechnologically relevant conditions, i.e. with high cell density and high aeration. Analytical boundary conditions for reliable analysis were determined, and perturbations in C-, N- or O- availability had marked impact on the cytosolic NADH/NAD+ ratio. NAD redox dynamics could be manipulated in lines inducibly expressing a water-forming NADH oxidase as a synthetic reductant sink. The establishment of Peredox-mCherry in U. maydis and the analysis of NAD redox dynamics provides a versatile methodology for the in vivo investigation of cellular metabolism, and contributes fundamental knowledge for rational design and optimization of biocatalysts.

Promoters from the itaconate cluster of Ustilago maydis are induced by nitrogen depletion.

BACKGROUND: Ustilago maydis is known for its natural potential to produce a broad range of valuable chemicals, such as itaconate, from both industrial carbon waste streams and renewable biomass. Production of itaconate, and many other secondary metabolites, is induced by nitrogen limitation in U. maydis. The clustered genes responsible for itaconate production have recently been identified, enabling the development of new expression tools that are compatible with biotechnological processes. RESULTS: Here we report on the investigation of two of the native promoters, P tad1 and P mtt1 , from the itaconate cluster of U. maydis MB215. For both promoters the specific activation upon nitrogen limitation, which is known to be the trigger for itaconate production in Ustilago, could be demonstrated by gfp expression. The promoters cover a broad range of expression levels, especially when combined with the possibility to create single- and multicopy construct integration events. In addition, these reporter constructs enable a functional characterization of gene induction patterns associated with itaconate production. CONCLUSIONS: The promoters are well suited to induce gene expression in response to nitrogen limitation, coupled to the itaconate production phase, which contributes towards the further improvement of organic acid production with Ustilago.

Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid production.

Ustilago trichophora RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain U. trichophora TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species. Here we investigate and adapt existing molecular tools from U. maydis for use in U. trichophora. Selection markers from U. maydis that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in U. trichophora. A plasmid was constructed containing the ip-locus of U. trichophora RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (mdh1, mdh2), and two malate transporters (ssu1, ssu2) was possible in U. trichophora TZ1 under control of the strong P etef promoter. Overexpression of mdh1, mdh2, ssu1, and ssu2 increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120 g L-1. In bioreactor cultivations of U. trichophora TZ1 P etefssu2 and U. trichophora TZ1 P etefmdh2 a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain U. trichophora TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134 g L-1 malate and biomass (73 g L-1), succinate (20 g L-1), CO2 (17 g L-1), and α-ketoglutarate (8 g L-1) as main by-products. These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation.

Efficient itaconic acid production from glycerol with Ustilago vetiveriae TZ1.

BACKGROUND: The family of Ustilaginaceae is known for their capability to naturally produce industrially valuable chemicals from different carbon sources. Recently, several Ustilaginaceae were reported to produce organic acids from glycerol, which is the main side stream in biodiesel production. RESULTS: In this study, we present Ustilago vetiveriae as new production organism for itaconate synthesis from glycerol. In a screening of 126 Ustilaginaceae, this organism reached one of the highest titers for itaconate combined with a high-glycerol uptake rate. By adaptive laboratory evolution, the production characteristics of this strain could be improved. Further medium optimization with the best single colony, U. vetiveriae TZ1, in 24-deep well plates resulted in a maximal itaconate titer of 34.7 ± 2.5 g L-1 produced at a rate of 0.09 ± 0.01 g L-1 h-1 from 196 g L-1 glycerol. Simultaneously, this strain produced 46.2 ± 1.4 g L-1 malate at a rate of 0.12 ± 0.00 g L-1 h-1. Due to product inhibition, the itaconate titer in NaOH-titrated bioreactor cultivations was lower (24 g L-1). Notably, an acidic pH value of 5.5 resulted in decreased itaconate production, however, completely abolishing malate production. Overexpression of ria1 or mtt1, encoding a transcriptional regulator and mitochondrial transporter, respectively, from the itaconate cluster of U. maydis resulted in a 2.0-fold (ria1) and 1.5-fold (mtt1) higher itaconate titer in comparison to the wild-type strain, simultaneously reducing malate production by 75 and 41%, respectively. CONCLUSIONS: The observed production properties of U. vetiveriae TZ1 make this strain a promising candidate for microbial itaconate production. The outcome of the overexpression experiments, which resulted in reduced malate production in favor of an increased itaconate titer, clearly strengthens its potential for industrial itaconate production from glycerol as major side stream of biodiesel production.

Draft Genome Sequences of Itaconate-Producing Ustilaginaceae.

Some smut fungi of the family Ustilaginaceae produce itaconate from glucose. De novo genome sequencing of nine itaconate-producing Ustilaginaceae revealed genome sizes between 19 and 25 Mbp. Comparison to the itaconate cluster of U. maydis MB215 revealed all essential genes for itaconate production contributing to metabolic engineering for improving itaconate production.

Draft Genome Sequence of Ustilago trichophora RK089, a Promising Malic Acid Producer.

The basidiomycetous smut fungus Ustilago trichophora RK089 produces malate from glycerol. De novo genome sequencing revealed a 20.7-Mbp genome (301 gap-closed contigs, 246 scaffolds). A comparison to the genome of Ustilago maydis 521 revealed all essential genes for malate production from glycerol contributing to metabolic engineering for improving malate production.

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.