Thiemo Zambanini

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 Petef 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 Petefssu2 and U. trichophora TZ1 Petefmdh2 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.

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

ABSTRACT 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.

Draft Genome Sequences of Itaconate-Producing Ustilaginaceae

ABSTRACT 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.

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.

Microbial challenges for domestic heating oil storage tanks

Microbial growth on hydrocarbons is common in nature and used in bioremediation of contaminated sites, whereas in fuel storage tanks this phenomenon can affect the stability of the fuel and the tank. The impact of microbial growth and produced metabolites on materials, which are used in the construction of storage tanks, were analyzed. In contrast to metal tank components, polymeric materials did not affect or were influenced by microorganisms. Zinc was highly corroded by microbial growth, most likely due to the formation of organic acids that were produced during microbial growth on hydrocarbons. A contaminated water phase in a storage tank of a heating system was simulated with a self‐constructed pump test bench. Microbial growth began in the water phase of the storage tank and microbes were distributed throughout the tank system, through water‐in‐oil microemulsions. No microbial growth was observed in oil that was previously contaminated, indicating that essential nutrients had been depleted. The identification and removal of these essential nutrients from fuels could minimize or prevent microbial contamination. The results are discussed with regard to developing recommendations for the design and operation of domestic heating oil storage tanks to lower the risk of technical failure due to microbial contamination.

Malatproduktion aus Rohglycerin mit Ustilago

The use of biodiesel derived crude glycerol for the production of value added chemicals has been discussed frequently. Current glycerol-based microbial production process often suffers from low rates, titers, and yields, hindering their industrial application. Here we report on the identification and optimization of Ustilago trichophora as promising novel production organism for malic acid from glycerol. All optimization steps presented avoid metabolic engineering resulting in a non-GMO organism.

What can we do with smut? Organic acid production from glycerol with Ustilaginaceae

economic and ecological advantage. Thus, the overall aim of this thesis was to establish a production process for organic acids from glycerol with Ustilaginaceae. In a screening of 126 Ustilaginaceae, two candidates for organic acid production were found Ustilago trichophora for malic acid and U. vetiveriae for itaconic acid. Glycerol uptake and growth rate of both strains were improved by adaptive laboratory evolution. Selection of the best growing colony for each strain and medium and process optimization drastically improved the production.