Thomas Brück

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Organization: Technical University of Munich , Germany

Department: 1 Fachgebiet fur Industrielle Biokatalyse, Zentrum fur Weiße Biotechnologie, Department fur Chemie

Title: Professor(PhD)

Chemicals
References

The effects of TORC signal interference on lipogenesis in the oleaginous yeast Trichosporon oleaginosus

Co-reporter:Felix Bracharz;Veronika Redai;Kathrin Bach;Farah Qoura

BMC Biotechnology 2017 Volume 17( Issue 1) pp:27

Publication Date(Web):07 March 2017

DOI:10.1186/s12896-017-0348-3

Oleaginous organisms are a promising, renewable source of single cell oil. Lipid accumulation is mainly induced by limitation of nutrients such as nitrogen, phosphorus or sulfur. The oleaginous yeast Trichosporon oleaginosus accumulates up to 70% w/w lipid under nitrogen stress, while cultivation in non-limiting media only yields 9% w/w lipid. Uncoupling growth from lipid accumulation is key for the industrial process applicability of oleaginous yeasts. This study evaluates the effects of rapamycin on TOR specific signaling pathways associated with lipogenesis in Trichosporon oleaginosus for the first time.Supplementation of rapamycin to nutrient rich cultivation medium led to an increase in lipid yield of up to 38% g/L. This effect plateaued at 40 μM rapamycin. Interestingly, the fatty acid spectrum resembled that observed with cultivation under nitrogen limitation. Significant changes in growth characteristics included a 19% increase in maximum cell density and a 12% higher maximum growth rate. T. oleaginosus only has one Tor gene much like the oleaginous yeast Rhodosporidium toruloides. Consequently, we analyzed the effect of rapamycin on T. oleaginosus specific TORC signaling using bioinformatic methodologies.We confirm, that target of rapamycin complex 1 (TORC1) is involved in control of lipid production and cell proliferation in T. oleaginosus and present a homology based signaling network. Signaling of lipid induction by TORC1 and response to carbon depletion to this complex appear to be conserved, whereas response to nitrogen limitation and autophagy are not. This work serves as a basis for further investigation regarding the control and induction of lipid accumulation in oil yeasts.

Open thin-layer cascade reactors for saline microalgae production evaluated in a physically simulated Mediterranean summer climate

Co-reporter:A.C. Apel, C.E. Pfaffinger, N. Basedahl, N. Mittwollen, J. Göbel, J. Sauter, T. Brück, D. Weuster-Botz

Algal Research 2017 Volume 25(Volume 25) pp:

Publication Date(Web):1 July 2017

DOI:10.1016/j.algal.2017.06.004

•An open photobioreactor for mass production of saline microalgae was designed.•Reactors were evaluated with a realistic physical climate simulation technology.•Nannochloropsis salina batch cultivation in a Mediterranean summer climate•Productivity, efficiency, growth rate, and biomass density exceeded literature data.•Pond liner channels enable inexpensive thin-layer cascade reactors on a large scale.While microalgae hold the promise for conversion of sunlight and CO2 to a wide variety of products, the economics of algae processes are still debatable. We have designed an open thin-layer cascade photobioreactor for high-cell density cultivation of saline microalgae to advance economic microalgae mass production. Pilot-scale reactors with a surface area of up to 8 m2 (cultivation volume 50–140 L) were constructed and evaluated using a dynamic climate simulation technology (light, air temperature and humidity) integrating natural sunlight and multi-color LED arrays for a highly realistic reproduction of the sunlight spectrum. Batch processes with Nannochloropsis salina were performed in these reactors in the physically simulated Mediterranean summer climate of Almería, Spain – an ideal location for outdoor microalgae cultivation. Two reactor variants were examined: one with a smooth but expensive rigid channel made of polyethylene sheets, and one with a more uneven but significantly less expensive channel made of pond liner. Maximal intra-day growth rates of 1.9 d−1 were observed at a cell density of 1–3 g L−1. The maximal cell density of 50 g L−1 was obtained within 25 days. These high growth rates and cell densities markedly exceed literature data. No difference in growth between the channel variants was observed. This suggests that cost-efficient large-scale thin-layer cascade reactors with inexpensive pond liner channels are feasible. The high cell density allows a reduction of harvesting cost. Optimal process conditions were identified by analyzing the batch and daily economic bioprocess metrics: At a cell density of 17 g L−1, an areal biomass productivity of 25 g m−2 d−1 (volumetric productivity 4 g L−1 d−1) and a photosynthetic conversion efficiency of 4.6% were observed. The reactor design is discussed in detail to encourage further advancement of thin-layer algal cultivation technology.Download high-res image (300KB)Download full-size image

Genetic engineering and production of modified fatty acids by the non-conventional oleaginous yeast Trichosporon oleaginosus ATCC 20509

Co-reporter:Christian Görner, Veronika Redai, Felix Bracharz, Patrick Schrepfer, Daniel Garbe and Thomas Brück

Green Chemistry 2016 vol. 18(Issue 7) pp:2037-2046

Publication Date(Web):25 Nov 2015

DOI:10.1039/C5GC01767J

The oleaginous yeast Trichosporon oleaginosus ATCC 20509 can accumulate up to 70% (w/DCW) triglycerides when cultivated on chemically diverse agricultural or food waste streams. In contrast to other lipogenic yeasts T. oleaginosus is able to efficiently convert constituents of hemicellulose and chitin hydrolysates into lipids. This study focused on establishing the genetic accessibility of T. oleaginosus aimed at manipulating lipid biosynthesis in order to generate high value lipids from waste streams. We demonstrate the first transformation protocol for T. oleaginosus based on Agrobacterium tumefaciens. Strong heterologous gene expression of a codon optimized YFP reporter protein was achieved using the constitutive promotor from the endogenous glyceraldehyde-3-phosphate dehydrogenase gene. Subsequently, we evaluated the ability of T. oleaginosus to generate non-natural fatty acid profiles by heterologous expression of several fatty acid modifying enzymes. De novo lipid generation of these recombinant strains was evaluated on diverse carbon sources. Compared to the wild type, recombinant yeast strains showed an increase of α-linolenic acid production from 2.8% to 21% with respect to the total cellular fatty acid content (TFA). Further, we designed yeast strains able to generate the non-native, polyunsaturated very long chain fatty acids eicosatrienoic (16% TFA) and eicosadienoic acid (9% TFA), respectively. Alternatively, T. oleaginosus was engineered to produce the non-native (E-10, Z-12) conjugated linoleic acid, which was generated up to 2.6% TFA. This work demonstrates, that T. oleaginosus ATCC 20509 can be used as versatile biotechnology platform to transform industrial waste streams into designed, high value fatty acids.

Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases

Co-reporter:Christian Goerner;Patrick Schrepfer;Michael Hertel;Alexander Buettner;Frank Wallrapp;Jeaphianne van Rijn;Wolfgang Eisenreich;Volker Sieber;Robert Kourist

PNAS 2016 Volume 113 (Issue 8 ) pp:E958-E967

Publication Date(Web):2016-02-23

DOI:10.1073/pnas.1519680113

Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure–function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.

Application of light-emitting diodes (LEDs) in cultivation of phototrophic microalgae: current state and perspectives

Co-reporter:M. Glemser;M. Heining;J. Schmidt;A. Becker

Applied Microbiology and Biotechnology 2016 Volume 100( Issue 3) pp:1077-1088

Publication Date(Web):2016 February

DOI:10.1007/s00253-015-7144-6

The quality and regulation of the incident light is crucial in microalgae cultivation processes. Depending on wavelength, spectrum, and intensity, growth characteristics and biochemical composition of these organisms vary. With mainly fluorescent lamps (FL) used previously for illumination, such variabilities could not be studied adequately due to their broad emission spectrum. In contrast, light-emitting diodes (LEDs) emit a very narrow wavelength band and enable flexible photobioreactor designs due to their small size. This review provides a condensed overview on the application of LEDs in microalgal cultivation processes. It summarizes the current availability and applicability of LED technologies as an illumination source for research-focused photobioreactor systems. A particular focus is the use of narrow-wavelength LEDs to address fundamental as well as applied aspects of light color on algae biomass and value-added compound formation. In this respect, the application of internal and external illumination systems is reviewed together with trends in the industrial use of LED systems to intensify algae process efficiency.

Detailed StructureFunction Correlations of Bacillus subtilis Acetolactate Synthase

Co-reporter:Dr. Bettina Sommer;Dr. Holger von Moeller;Martina Haack;Dr. Farah Qoura;Clemens Langner;Dr. Gleb Bourenkov;Dr. Daniel Garbe;Dr. Bernhard Loll; Dr. Thomas Brück

ChemBioChem 2015 Volume 16( Issue 1) pp:110-118

Publication Date(Web):

DOI:10.1002/cbic.201402541

Abstract

Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.1 Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.2–6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg²⁺ as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg²⁺ and in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.

Nachhaltige Bausteine fr die Kunststoff-Herstellung : Biomasse-basierte Lipide

Co-reporter:Daniel Garbe;Steven Reiße ; Dr. Thomas Brück

Chemie in unserer Zeit 2014 Volume 48( Issue 4) pp:284-295

Publication Date(Web):

DOI:10.1002/ciuz.201400673

Abstract

Die Produktion biobasierter Hochleistungskunststoffe wie Polyamide nutzt Pflanzenöle als Rohstoffquelle. Fortschritte in chemischen und biotechnologischen Katalyse-Verfahren ermöglichen heute die Konversion von Pflanzenölen in maßgeschneiderte Bausteine für die Polymerproduktion. Schon heute haben biobasierte Polymer-Synthesebausteine äquivalente chemische und physikalische Eigenschaften als auch vergleichbare Kostenstrukturen wie petrochemische Synthesegrundstoffe. Dies ermöglicht die Integration Biomasse-basierter Rohstoffströme in bestehende Prozesskonfigurationen im industriellen Maßstab. Jedoch wird erst die effiziente Nutzung von Synergien zwischen chemischen und biotechnologischen Verfahren ein nachhaltiges und umweltschonendes Prozessdesign in Zukunft erlauben. Um langfristig Bio-Öle für die chemische Industrie bereitstellen zu können, ohne signifikant in die Nahrungsmittelproduktion einzugreifen, müssen neue Verfahren für die Produktion mikrobieller Öle auf Basis von Biomasse-Reststoffströmen entwickelt werden.

Plant oils are currently the principle resource for the production of bio-based, high performance polymers, such as polyamides. This process is facilitated by giant strides in chemical catalysis and biotechnology, which allows conversion of vegetable oils in “drop-in” chemical building blocks. These bio-based polymer building blocks have equivalent chemical and physical properties as well as similar cost structures compared to conventional petrochemical synthesis feedstock. This allows integration of bio-based resources into industrial production processes without significant adaptations in logistics or process configuration. However, only use of synergies between chemical and biotechnological unit operations will in future provide for sustainable and eco-efficient process designs. To allow sustainable supply of bio-oils to a growing chemical industry without a significant impact on food production demands development of alternative bio-oil sourcing strategies. In this respect the development of processes for the production of microbial oils, which have equivalent chemical properties to their plant counterparts is imperative. One leading option is the biotechnological conversion of agricultural and food waste streams into microbial oils by combining enzymatic hydrolysis and fermentative production using oleaginous organisms, such as yeasts.

Production of Macrocyclic Sesqui- and Diterpenes in Heterologous Microbial Hosts: A Systems Approach to Harness Natures Molecular Diversity

Co-reporter:Dr. Thomas Brück;Dr. Robert Kourist;Dr. Bernhard Loll

ChemCatChem 2014 Volume 6( Issue 5) pp:1142-1165

Publication Date(Web):

DOI:10.1002/cctc.201300733

Abstract

Macrocyclic sesqui- (C15) and diterpenes (C20) are a functionally diverse group of natural products with versatile bioactivities encompassing anticancer, antimicrobial and insecticidal agents. Structural complexity prevents economically efficient total synthesis of these higher terpenoids. Heterologous production in recombinant whole-cell biocatalysts is an emerging alternative. Conventional cell systems (i.e., Escherichia coli and Saccharomyces cerevisiae) frequently suffer from low volumetric yields. However, recent combinations of metabolic, enzyme and process engineering in conjunction with systems biology allow significant improvements towards economically viable processes. This Review analyzes research trends in the dynamic fields of terpene-centered microbial cell systems and enzyme engineering. An outlook is given on emerging microbial hosts, which may simplify cellular engineering towards higher product titers.

Targeted Engineering of Cyclooctat-9-en-7-ol Synthase: A Stereospecific Access to Two New Non-natural Fusicoccane-Type Diterpenes

Co-reporter:Christian Görner;Ina Häuslein;Patrick Schrepfer;Dr. Wolfgang Eisenreich;Dr. Thomas Brück

ChemCatChem 2013 Volume 5( Issue 11) pp:3289-3298

Publication Date(Web):

DOI:10.1002/cctc.201300285

Abstract

The structural diversity of bioactive diterpenes is due to variations in their macrocyclic carbon skeletons. The chemical synthesis of these macrocycles is challenging. However, the bacterial diterpene synthase cyclooctat-9-en-7-ol synthase (CotB2) generates a complex macrocycle in a single step with geranylgeranyl diphosphate as an aliphatic substrate. This study investigates the catalytic mechanisms of the native and mutant CotB2, with a focus on identifying new carbon macrocycles. The combination of in silico modelling, targeted diterpene cyclase engineering and structural elucidation by using GC–MS, HRMS and NMR analysis resulted in the identification of new terpene olefins. CotB2 mutants produced two new non-natural fusicoccane-type macrocycles with potential bioactivities and the monocyclic compound cembrene. The observed product pattern allowed insights into the mechanistic features of CotB2. Applied strategies enable new consolidated synthesis of natural and non-natural terpenoid bioactives.

Characterization of a new, recombinant thermo-active subtilisin-like serine protease derived from Shewanella arctica

Co-reporter:Farah Qoura, Elias Kassab, Steven Reiße, Garabed Antranikian, Thomas Brueck

Journal of Molecular Catalysis B: Enzymatic (June 2015) Volume 116() pp:16-23

Publication Date(Web):1 June 2015

DOI:10.1016/j.molcatb.2015.02.015

•The hydrolase enzyme family proteases comprise the commercially most important activities with application in the food/feed, cosmetics, pharmaceutical and fine chemical sector.•We have identified and characterized a new, subtilisin-like serine protease from the psychrotophic eubacterium Shewanella arctica.•The recombinant enzyme is active over a very broad temperature (T: 0–80 °C) and pH (pH 6–10) range, with optimal proteolytic activity at 60 °C and pH 8 respectively.•The enzyme exhibits an extremely long half-life of 45 h and 1.5 h at 40 °C and 70 °C, respectively.•The enzyme tolerates the presence of several metal ions and detergents without affecting activity.Proteases represent the commercially most relevant enzyme family with applications in the chemical, cosmetic and pharmaceutical industry. For the personal care industry, both cold-active and thermo-active protease variants are of particular interest for detergent formulation. We have identified a gene sequence encoding a putative subtilisin-like serine protease in a gene library of the psychrophilic bacterium Shewanella arctica (DSM 16509, JCM 14208). The recombinant protein (67 kDa, 644 amino acids) product was flanked by 4.4 kDa (38 a.a.) signal sequence. Initial in-silico sequence analysis and protein modelling revealed the dominant alpha helical structural features embedding the catalytic residues Asp-180, His-213 and Ser-364, which form the canonical catalytic triad of subtilisin-like serine proteases. Further, in the N-terminal region a subtilisin-N domain was detected between residues Asp-55 and Thr-135. Phylogenetic analysis indicated that this enzyme can be classified as subtilisin-like serine protease with 73% amino acids identity to its closest characterized relative, the putative protease of Pseudoaltermonas sp. This is the first report of a detailed biochemical characterization of Shewanella spp. derived protease activity. The recombinant protein (28.18 mg/L), termed SaP, was purified to homogeneity from a crude E. coli lysate with an activity of 0.52 U/mg. Surprisingly, although derived from psychrophilic bacteria, SaP is active over a very broad temperature (T = 0–80 °C) and pH (pH 6–10) range, with optimal proteolytic activity at 60 °C and pH 8. The unique enzyme displayed an activity half-life of 45 h and 1.5 h at 40 °C and 70 °C, respectively. The specific activity of SaP (Kcat/Km: 71.14 s−1 M−1) toward the model substrate casein is comparable to other prokaryotic serine proteases. The enzyme activity was not affected by various metal ions or non-specific protease inhibitors, which positions SaP for industrial application operating over a broad temperature range, such as household detergent formulations.Download full-size image

Identification and optimization of a novel thermo- and solvent stable ketol-acid reductoisomerase for cell free isobutanol biosynthesis

Co-reporter:Steven Reiße, Daniel Garbe, Thomas Brück

Biochimie (January 2015) Volume 108() pp:76-84

Publication Date(Web):January 2015

DOI:10.1016/j.biochi.2014.10.024

Identification and characterization of a highly thermostable crotonase from Meiothermus ruber

Co-reporter:Steven Reiße, Daniel Garbe, Thomas Brück

Journal of Molecular Catalysis B: Enzymatic (February 2015) Volume 112() pp:40-44

Publication Date(Web):1 February 2015

DOI:10.1016/j.molcatb.2014.11.011

•Genome mining allowed identification of a new crotonase.•Meiothermus ruber crotonase is highly thermostable.•Outstanding extended half-life over 1 month at 50 °C.Butanol is deemed a second generation biofuel due to its enhanced energy content and improved hydrophobicity compared to ethanol. The state of the art production is the Clostridia based anaerobic acetone, butanol and ethanol (ABE) fermentation process. However, the classical ABE fermentation is capped at 2% (v/v) butanol yield, due to end-product toxicity effects. By contrast, cell-free bio-butanol production systems based on designed enzyme cascades hold the promise of higher butanol yields and simplified end-product recovery. Crotonases catalyze the reversible hydration of crotonyl-CoA and are key enzymes in butanol biosynthesis. In this study, we present the isolation, heterologous expression and characterization of a new highly thermostable crotonase (Crt) derived from the bacterium Meiothermus ruber (Mr). Mr-Crt displays a broad activity range of 50–70 °C, with optimal reactivity at pH 7.0 and 55 °C. Moreover, Mr-Crt displays an extended half-life of over 1 month (IT50 (50 °C) = 743 ± 0.7 h) at elevated temperatures. Robust enzyme activities, such as Mr-Crt, with high temperature and solvent tolerance will ultimately contribute to an improved cell-free butanol process.Download full-size image

Characterization of a highly thermostable ß-hydroxybutyryl CoA dehydrogenase from Clostridium acetobutylicum ATCC 824

Co-reporter:Bettina Sommer, Daniel Garbe, Patrick Schrepfer, Thomas Brück

Journal of Molecular Catalysis B: Enzymatic (30 December 2013) Volume 98() pp:138-144

Publication Date(Web):30 December 2013

DOI:10.1016/j.molcatb.2013.10.014

•Hbd from Clostridium acetobutylicum was structurally and biochemically characterized.•Hbd is highly thermostable with Topt of 65 °C and a half-life of 42 h at 70 °C.•Hbd is extremely stable against the industrial solvents ethanol, isobutanol, butanol.•pH dependence of the reversible acetoacetyl CoA oxidation was elucidated.•NAD+ was identified as an uncompetitive inhibitor of Hbd.Higher energy content and hydrophobicity make bio-based n-butanol a preferred building block for chemical and biofuels manufacturing. Butanol is obtained by Clostridium sp. based ABE fermentation process. While the ABE process is well understood, the enzyme systems involved have not been elucidated in detail. The important enzyme ß-hydroxybutyryl CoA dehydrogenase from Clostridium acetobutylicum ATCC 824 (Hbd) was purified and characterized. Surprisingly, Hbd shows extremely high temperature (T > 60 °C), pH (4–11) and solvent (1-butanol, isobutanol, ethanol) stability. Hbd catalyzes acetoacetyl CoA hydration to ß-hydroxybutyryl CoA up to pH 9.5, where the reaction is reversed. Substrate (acacCoA, ß-hbCoA) and cofactor (NADH, NAD+, NADPH and NADP+) specificities were determined. We identified NAD+ as an uncompetitive inhibitor. Identification of process relevant enzymes such as Hbd is key to optimize butanol production via cellular or cell-free enzymatic systems.Download full-size image