Online evaluation of the metabolic activity of Ustilago maydis on (poly)galacturonic acid

Müller, Markus Jan; Stachurski, Sarah; Stoffels, Peter; Schipper, Kerstin; Feldbrügge, Michael; Büchs, Jochen

doi:10.1186/s13036-018-0128-1

Cited by 28 publications

(17 citation statements)

References 68 publications

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“…Resistance marker recycling allows the generation of marker free production strains as well as the generation of multiple gene deletions (Khrunyk et al, 2010;Terfrüchte et al, 2018). Importantly, online bioengineering procedures for fermentation of U. maydis are already implemented (Kreyenschulte et al, 2015;Müller et al, 2018).…”

Section: Production Of Plant and Fungal Sesquiterpenoids In U Maydismentioning

confidence: 99%

Ustilago maydis Serves as a Novel Production Host for the Synthesis of Plant and Fungal Sesquiterpenoids

et al. 2020

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Sesquiterpenoids are important secondary metabolites with various pharma-and nutraceutical properties. In particular, higher basidiomycetes possess a versatile biosynthetic repertoire for these bioactive compounds. To date, only a few microbial production systems for fungal sesquiterpenoids have been established. Here, we introduce Ustilago maydis as a novel production host. This model fungus is a close relative of higher basidiomycetes. It offers the advantage of metabolic compatibility and potential tolerance for substances toxic to other microorganisms. We successfully implemented a heterologous pathway to produce the carotenoid lycopene that served as a straightforward read-out for precursor pathway engineering. Overexpressing genes encoding enzymes of the mevalonate pathway resulted in increased lycopene levels. Verifying the subcellular localization of the relevant enzymes revealed that initial metabolic reactions might take place in peroxisomes: despite the absence of a canonical peroxisomal targeting sequence, acetyl-CoA C-acetyltransferase Aat1 localized to peroxisomes. By expressing the plant (+)-valencene synthase CnVS and the basidiomycete sesquiterpenoid synthase Cop6, we succeeded in producing (+)valencene and α-cuprenene, respectively. Importantly, the fungal compound yielded about tenfold higher titers in comparison to the plant substance. This proof of principle demonstrates that U. maydis can serve as promising novel chassis for the production of terpenoids.

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Section: Production Of Plant and Fungal Sesquiterpenoids In U Maydismentioning

confidence: 99%

Ustilago maydis Serves as a Novel Production Host for the Synthesis of Plant and Fungal Sesquiterpenoids

et al. 2020

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“…Here, the respiration rate of the fed-batch CBP was monitored online as a direct measure for microbial activity. As described in earlier studies (41,47), cellulose consumption and thereby suitable time points for feeding fresh substrate could be estimated online based on the cumulative oxygen consumption. This way, intermittent starvation of the culture could be prevented (Fig.…”

Section: In Uence Of Inoculation Timementioning

confidence: 99%

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

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et al. 2020

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Background: Itaconic acid is a bio-derived platform chemical with uses ranging from polymer synthesis to biofuel production. The efficient conversion of cellulosic waste streams into itaconic acid could thus enable the sustainable production of a variety of substitutes for fossil oil based products. However, the realization of such a process is currently hindered by an expensive conversion of cellulose into fermentable sugars. Here, we present the stepwise development of a fully consolidated bioprocess (CBP), which is capable to directly convert recalcitrant cellulose into itaconic acid without the need for separate cellulose hydrolysis including the application of commercial cellulases. The process is based on a synthetic microbial consortium of the cellulase producer Trichoderma reesei and the itaconic acid producing yeast Ustilago maydis. Results: The efficiency of the process was compared to a simultaneous hydrolysis and fermentation setup (SSF). Because of the additional substrate consumption of T. reesei in the CBP, the itaconic acid yield was significantly lower in the CBP than in the SSF. In order to increase yield and productivity of itaconic acid in the CBP, the population dynamics was manipulated by varying the inoculation delay between T. reesei and U. maydis. Surprisingly, neither inoculation delay nor inoculation density significantly affected the population development or the CBP performance. Instead, the substrate availability was the most important parameter. U. maydis was only able to grow and to produce itaconic acid when the cellulose concentration and thus, the sugar supply rate, was high. Finally, the metabolic processes during fed-batch CBP were analyzed in depth by online respiration measurements. A method was developed to estimate cellulose consumption, itaconic acid formation as well as the actual itaconic acid production yield online. Again, substrate availability was the key factor also controlling itaconic acid yield. In summary, an itaconic acid titer of 34 g/L with a total productivity of up to 0.07 g/L/h and a yield of 0.16 g/g could be reached during fed-batch cultivation. Conclusion: This study demonstrates the feasibility of consortium based CBP for itaconic acid production and also lays the fundament for the development and improvement of similar microbial consortia for cellulose based organic acid production.

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“…This includes itaconic acid as a chemical platform molecule for biofuels, ustilagic acid and mannosylerythritol lipids (MEL) as biosurfactants, and various antibody formats as valuable proteins ( Teichmann et al, 2010 ; Feldbrügge et al, 2013 ; Sarkari et al, 2014 ; Terfrüchte et al, 2017 ; Becker et al, 2020 ). Furthermore, strains were generated to utilise cellobiose, xylan and polygalacturonic acid as a carbon source in the yeast phase, so that plant cell wall components including pectin can be used as starting point for sustainable production ( Geiser et al, 2016 ; Müller et al, 2018 ; Stoffels et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Establishing Polycistronic Expression in the Model Microorganism Ustilago maydis

et al. 2020

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Eukaryotic microorganisms use monocistronic mRNAs to encode proteins. For synthetic biological approaches like metabolic engineering, precise co-expression of several proteins in space and time is advantageous. A straightforward approach is the application of viral 2A peptides to design synthetic polycistronic mRNAs in eukaryotes. During translation of these peptides the ribosome stalls, the peptide chain is released and the ribosome resumes translation. Thus, two independent polypeptide chains can be encoded from a single mRNA when a 2A peptide sequence is placed inbetween the two open reading frames. Here, we establish such a system in the well-studied model microorganism Ustilago maydis . Using two fluorescence reporter proteins, we compared the activity of five viral 2A peptides. Their activity was evaluated in vivo using fluorescence microscopy and validated using fluorescence resonance energy transfer (FRET). Activity ranged from 20 to 100% and the best performing 2A peptide was P2A from porcine teschovirus-1. As proof of principle, we followed regulated gene expression efficiently over time and synthesised a tri-cistronic mRNA encoding biosynthetic enzymes to produce mannosylerythritol lipids (MELs). In essence, we evaluated 2A peptides in vivo and demonstrated the applicability of 2A peptide technology for U. maydis in basic and applied science.

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Online evaluation of the metabolic activity of Ustilago maydis on (poly)galacturonic acid

Cited by 28 publications

References 68 publications

Ustilago maydis Serves as a Novel Production Host for the Synthesis of Plant and Fungal Sesquiterpenoids

Ustilago maydis Serves as a Novel Production Host for the Synthesis of Plant and Fungal Sesquiterpenoids

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

Establishing Polycistronic Expression in the Model Microorganism Ustilago maydis

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