Andreas Schmid

Helmholtz-Zentrum für Umweltforschung, Leipzig, Saxony, Germany

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Publications (163)680.03 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Recent environmental economic developments generate a need for sustainable and cost-effective (microbial) processes for the production of high-volume, low-priced bulk chemicals. As an example, n-Butanol has, as a 2nd generation biofuel, beneficial characteristics compared to ethanol in liquid transportation fuel applications. The industrial revival of the classic n-butanol (ABE) fermentation requires process and strain engineering solutions for overcoming the main process limitations: product toxicity and low space-time yield (STY). Reaction intensification on the biocatalyst, fermentation, and bioprocess level can be based on economic and ecologic evaluations using quantifiable constraints. This review describes the means of process intensification for biotechnological processes. A quantitative approach is then used for the comparison of the massive literature on n-butanol fermentation. A comprehensive literature study – including key fermentation performance parameters – is presented and the results are visualized using the window of operation methodology. The comparison allowed the identification of the key constraints high cell densities, high strain stability, high specific production rate, cheap in situ product removal (ISPR), high n-butanol tolerance to operate ISPR efficiently, and cheap carbon source. It can thus be used as a guideline for the bioengineer during the combined biocatalyst, fermentation, and bioprocess development and intensification.This article is protected by copyright. All rights reserved
    Engineering in Life Sciences 11/2014; · 1.63 Impact Factor
  • Christian Dusny, Andreas Schmid
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    ABSTRACT: Life is based on the cell as the elementary replicative and self-sustaining biological unit. Each single cell constitutes an independent and highly dynamic system with a remarkable individuality in a multitude of physiological traits and responses to environmental fluctuations. However, with traditional population-based cultivation set-ups, it is not possible to decouple inherent stochastic processes and extracellular contributions to phenotypic individuality for two central reasons: the lack of environmental control and the occlusion of single-cell dynamics by the population average. With microfluidic single-cell analysis as a new cell assay format, these issues can now be addressed, enabling cultivation and time-resolved analysis of single cells in precisely manipulable extracellular environments beyond the bulk. In this article, we explore the interplay of cellular physiology and environment at a single-cell level. We review biological basics that govern the functional state of the cell and put them in context with physical fundamentals that shape the extracellular environment. Furthermore, the significance of single-cell growth rates as pivotal descriptors for global cellular physiology is discussed and highlighted by selected studies. These examples illustrate the unique opportunities of microfluidic single-cell cultivation in combination with growth rate analysis, addressing questions of fundamental bio(techno)logical interest.
    Environmental Microbiology 10/2014; · 6.24 Impact Factor
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    ABSTRACT: The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, either interesting as substrate or product, or as second phase for in situ product removal and tool to control multistep biocatalysis. Solvent tolerant bacteria, especially Pseudomonas strains are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120 and adaptive solvent tolerance was proven by cultivation in the presence of 1% (v/v) toluene. Deletion of ttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent tolerant mutants of P. taiwanensis VLB120 and VLB120ΔC. Interestingly, the increased amount of solvent efflux pumps did not only enhance growth in the presence of toluene and styrene, but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Maximum specific epoxidation activities of P. taiwanensis VLB120ΔCΔttgV doubled to 67 U/gCDW compared to the parent strain P. taiwanensis VLB120ΔC. This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds, but can also be used as tools to improve redox biocatalysis involving organic solvents.
    Applied and environmental microbiology. 08/2014;
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    ABSTRACT: Catalytic biofilms minimize reactant toxicity and maximize biocatalyst stability in selective transformations of chemicals to value-added products in continuous processes. The scaling up of such catalytic biofilm processes is challenging, due to fluidic and biological parameters affording a special reactor design affecting process performance. A solid support membrane aerated biofilm reactor was optimized and scaled-up to yield gram amounts of (S)-styrene oxide, a toxic and instable high value chemical synthon. A sintered stainless steel membrane unit was identified as an optimal choice as biofilm substratum and for high oxygen mass transfer. A stable expanded PTFE (ePTFE) membrane was best suited for in situ substrate delivery and product extraction. For the verification of scalability, catalytic biofilms of Pseudomonas sp. strain VLB120ΔC produced (S)-styrene oxide to an average concentration of 390 mM in the organic phase per day (equivalent to 24.4 g Laq (-1) day(-1) ). This productivity was gained by efficiently using the catalyst with an excellent product yield on biomass of 13.6 gproduct gbiomass (-1) . This product yield on biomass is in the order of magnitude reported for other continuous systems based on artificially immobilized biocatalysts and is fulfilling the minimum requirements for industrial biocatalytic processes. Overall, 46 g of (S)-styrene oxide were produced and isolated (purity: 99%; ee: > 99.8%. yield: 30%). The productivity is in a similar range as in comparable small scale biofilm reactors highlighting the large potential of this methodology for continuous bioprocessing of bulk chemicals and biofuels.
    Biotechnology Journal 08/2014; · 3.71 Impact Factor
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    ABSTRACT: Microreactors provide higher mass transfer rates than do conventional batch reactors. A tube-in-tube microreactor was used for the NADH-dependent in vitro conversion of 2-hydroxybiphenyl to 3-phenylcatechol that was catalysed by 2-hydroxybiphenyl 3-monooxygenase. A biphasic reaction system allowed high substrate loadings, whereas the microreactor ensured excellent mass transfer rates between the organic and aqueous phases. Oxygen was supplied continuously by membrane aeration across the whole reaction compartment. The productivities achieved in the tube-in-tube microreactor were 38 times higher than those in previously described batch reactors and almost 4 times higher than for the same reaction in a microreactor in which aqueous, organic, and air phases were delivered through consecutive segments. This set-up is a promising concept for oxygen-dependent biocatalytic reactions in microreactors and is developing as a basis for applications in gram-scale organic biosyntheses.
    ChemCatChem 08/2014; · 5.18 Impact Factor
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    ABSTRACT: The efficient coupling of biotransformation steps to an existing fermentation pathway is an interesting strategy to expand the product portfolio of Corynebacterium glutamicum as whole-cell biocatalyst. This is especially challenging if the biotransformation step comprises a direct link to central metabolism, as in the case of α-ketoglutarate-dependent oxygenase catalysis. Aiming at trans-4-hydroxy-L-proline (Hyp) production from glucose in a minimal medium, the proline-4-hydroxylase gene from Dactylosporangium sp. strain RH1 was introduced into a proline-producing, isoleucine-bradytroph C. glutamicum strain. The production of proline was found to be induced by isoleucine limitation. Proline and Hyp production were found to depend differently on isoleucine limitation. Severe isoleucine limitation was shown to result in proline accumulation and low hydroxylation rates both in batch and continuous cultivation set-ups. The investigation of different steady states with various glucose:isoleucine molar ratios revealed that optimal conditions for Hyp production are met around a molar ratio of 46:1, where isoleucine limitation is sufficient to trigger proline production but the hydroxylation rate is high enough to convert the majority of formed proline to Hyp. A high cell-density fed-batch set-up was designed, capable of producing 7.1 g L−1 of Hyp from glucose in 23 h with 98.5% conversion of proline to Hyp. Reaction engineering, specifically the fine-tuning of the glucose:isoleucine concentration ratio, enabled control of the fermentation profile and thus the accumulation of the desired product Hyp from glucose in minimal and defined media. Biotechnol. Bioeng. © 2014 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 08/2014; · 4.16 Impact Factor
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    ABSTRACT: What defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway and the citric acid cycle. The prevalence of this definition of central metabolism is however equivocal without an experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between the steady states on alternative carbon sources. The approach is investigated in the model bacterium Pseudomonas putida with glucose, fructose and benzoate as carbon sources. The catabolic reactions involved in the initial uptake and metabolism of these substrates are expected to show a correlated change in gene expression and metabolic fluxes. However, there was no correlation for the reactions linking the twelve-biomass precursor molecules, indicating a regulation mechanism other than mRNA synthesis for central metabolism. This result substantiates evidence for a (re-) definition of central carbon metabolism including all reactions that are bound to tight regulation and transcriptional invariance. Contrary to expectations, the canonical Entner-Doudoroff and the EMP pathways sensu stricto are not a part of central carbon metabolism in P. putida, as they are not regulated differently to the aromatic degradation pathway. The regulatory analyses presented here provide leads on a qualitative basis to address the use of alternative carbon sources by de-regulation and over expression at the transcriptional level, while rate improvements in central carbon metabolism require careful adjustment of metabolite concentrations, as regulation resides to a large extent on post-translational and/or metabolic regulation.
    Applied and environmental microbiology. 06/2014;
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    ABSTRACT: The natural ability of Pseudomonas taiwanensis VLB120 to use xylose as sole carbon and energy source offers a high potential for sustainable industrial biotechnology. In general, three xylose assimilation routes are reported for bacteria. To elaborate the metabolic capacity of P. taiwanensis VLB120 and to identify potential targets for metabolic engineering, an in silico/in vivo experiment was designed, allowing for discrimination between these pathways. Kinetics of glucose and xylose degradation in P. taiwanensis VLB120 were determined and the underlying stoichiometry was investigated by genome-based metabolic modeling and tracer studies using stable isotope labeling. Additionally RT-qPCR experiments have been performed to link physiology to the genomic inventory. Based on in silico experiments a labeling strategy was developed, ensuring a measurable and unique (13) C-labeling distribution in proteinogenic amino acids for every possible distribution between the different xylose metabolization routes. A comparison to in vivo results allows the conclusion that xylose is metabolized by P. taiwanensis VLB120 via the Weimberg pathway. Transcriptomic and physiological studies point to the biotransformation of xylose to xylonate by glucose dehydrogenase. The kinetics of this enzyme are also responsible for the preference of glucose as carbon source by cells growing in the presence of glucose and xylose.
    Environmental Microbiology 06/2014; · 6.24 Impact Factor
  • Reto Ruinatscha, Katja Buehler, Andreas Schmid
    Journal of Molecular Catalysis B Enzymatic 05/2014; 103:100–105. · 2.82 Impact Factor
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    ABSTRACT: The oxyfunctionalization of unactivated CH bonds can selectively and efficiently be catalyzed by oxygenase-containing whole-cell biocatalysts. Recombinant Escherichia coli W3110 containing the alkane monooxygenase AlkBGT and the outer membrane protein AlkL from Pseudomonas putida GPo1 have been shown to efficiently catalyze the terminal oxyfunctionalization of renewable fatty acid methyl esters yielding bifunctional products of interest for polymer synthesis. In this study, AlkBGTL-containing E. coli W3110 is shown to catalyze the multistep conversion of dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to the acid, exhibiting Michaelis–Menten-type kinetics for each reaction step. In two-liquid phase biotransformations, the product formation pattern was found to be controlled by DAME availability. Supplying DAME as bulk organic phase led to accumulation of the terminal alcohol as the predominant product. Limiting DAME availability via application of bis(2-ethylhexyl)phthalate (BEHP) as organic carrier solvent enabled almost exclusive acid accumulation. Furthermore, utilization of BEHP enhanced catalyst stability by reducing toxic effects of substrate and products. A further shift towards the overoxidized products was achieved by co-expression of the gene encoding the alcohol dehydrogenase AlkJ, which was shown to catalyze efficient and irreversible alcohol to aldehyde oxidation in vivo. With DAME as organic phase, the aldehyde accumulated as main product using resting cells containing AlkBGT, AlkL, as well as AlkJ. This study highlights the versatility of whole-cell biocatalysis for synthesis of industrially relevant bifunctional building blocks and demonstrates how integrated reaction and catalyst engineering can be implemented to control product formation patterns in biocatalytic multistep reactions. Biotechnol. Bioeng. 2014;9999: 1–11. © 2014 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 05/2014; · 4.16 Impact Factor
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    ABSTRACT: The efficiency and productivity of cellular biocatalysts play a key role in the industrial synthesis of fine and bulk chemicals. This study focuses on optimizing the synthesis of (S)-limonene from glycerol and glucose as carbon sources using recombinant E. coli. The cyclic monoterpene limonene is extensively used in the fragrance, food, and cosmetic industries. Recently, limonene also gained interest as alternative jet fuel from biological origin. Key parameters that limit the (S)-limonene yield related to genetics, physiology, and reaction engineering were identified. The growth-dependent production of (S)-limonene was shown for the first time in minimal media. E. coli BL21 (DE3) was chosen as the preferred host strain as it showed low acetate formation, fast growth, and high productivity. A two-liquid phase fed-batch fermentation with glucose as sole carbon and energy source resulted in the formation of 700 mg Lorg (-1) (S)-limonene. Specific activities of 75 mU gcdw (-1) were reached, but decreased relatively quickly. The use of glycerol as carbon source resulted in a prolonged growth and production phase (specific activities of ≥50 mU gcdw (-1) ) leading to a final (S)-limonene concentration of 2,700 mg Lorg (-1) . Although geranyl diphosphate (GPP) synthase had a low solubility, its availability appeared not to limit (S)-limonene formation in vivo under the conditions investigated. GPP rerouting towards endogenous farnesyl diphosphate (FPP) formation did not limit (S)-limonene production either. The two-liquid phase fed-batch setup led to the highest monoterpene concentration obtained with a recombinant microbial biocatalyst to date.
    Biotechnology Journal 04/2014; · 3.71 Impact Factor
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    ABSTRACT: Biofilm reactors are often mass transfer limited due to excessive biofilm growth, impeding reactor performance. Fluidic conditions play a key role for biofilm structural development and subsequently for overall reactor performance. Continuous interfacial forces generated by aqueous-air segmented flow are controlling biofilm structure and diminish mass transfer limitations in biofilm microreactors. A simple three step method allows the formation of robust biofilms under aqueous-air segmented flow conditions: a first-generation biofilm is developing during single phase flow, followed by the introduction of air segments discarding most of the established biofilm. Finally, a second-generation, mature biofilm is formed in the presence of aqueous-air segments. Confocal laser scanning microscopy experiments revealed that the segmented flow supports the development of a robust biofilm. This mature biofilm is characterized by a three to four fold increase in growth rate, calculated from an increase in thickness, a faster spatial distribution (95% surface coverage in 24h), and a significantly more compact structure (roughness coefficient <1), as compared to biofilms grown under single phase flow conditions. The applicability of the concept in a segmented flow biofilm microreactor was demonstrated using the epoxidation of styrene to (S)-styrene oxide (ee > 99.8%) catalyzed by Pseudomonas sp. strain VLB120▵C cells in the mono-species biofilm. The limiting factor affecting reactor performance was oxygen transfer as the volumetric productivity rose from 11 to 46 g Ltube−1 day−1 after increasing the air flow rate. In summary, different interfacial forces can be applied for separating cell attachment and adaptation resulting in the development of a robust catalytic biofilm in continuous microreactors. Biotechnol. Bioeng. © 2014 Wiley Periodicals, Inc.
    Biotechnology and Bioengineering 04/2014; · 4.16 Impact Factor
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    ABSTRACT: Over the recent years the production of Ehrlich pathway derived chemicals was shown in a variety of hosts such as Escherichia coli, Corynebacterium glutamicum, and yeast. Exemplarily the production of isobutyric acid was demonstrated in Escherichia coli with remarkable titers and yields. However, these examples suffer from byproduct formation due to the fermentative growth mode of the respective organism. We aim at establishing a new aerobic, chassis for the synthesis of isobutyric acid and other interesting metabolites using Pseudomonas sp. strain VLB120, an obligate aerobe organism, as host strain. The overexpression of kivd, coding for a 2-ketoacid decarboxylase from Lactococcus lactis in Ps. sp. strain VLB120 enabled for the production of isobutyric acid and isobutanol via the valine synthesis route (Ehrlich pathway). This indicates the existence of chromosomally encoded alcohol and aldehyde dehydrogenases catalyzing the reduction and oxidation of isobutyraldehyde. In addition we showed that the strain possesses a complete pathway for isobutyric acid metabolization, channeling the compound via isobutyryl-CoA into valine degradation. Three key issues were addressed to allow and optimize isobutyric acid synthesis: i) minimizing isobutyric acid degradation by host intrinsic enzymes, ii) construction of suitable expression systems and iii) streamlining of central carbon metabolism finally leading to production of up to 26.8 +/- 1.5 mM isobutyric acid with a carbon yield of 0.12 +/- 0.01 g gglc-1. The combination of an increased flux towards isobutyric acid using a tailor-made expression system and the prevention of precursor and product degradation allowed efficient production of isobutyric acid in Ps. sp. strain VLB120. This will be the basis for the development of a continuous reaction process for this bulk chemicals.
    Microbial Cell Factories 01/2014; 13(1):2. · 3.31 Impact Factor
  • Journal of Molecular Catalysis B Enzymatic 01/2014; · 2.82 Impact Factor
  • Andreas Schmid
    Journal of Molecular Catalysis B Enzymatic 01/2014; · 2.82 Impact Factor
  • Nadine Ladkau, Andreas Schmid, Bruno Bühler
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    ABSTRACT: Whole-cell biocatalysis has emerged as an important tool for the synthesis of value-added fine and bulk chemicals as well as pharmaceuticals. Especially, the rapid development of recombinant DNA technologies resulted in a shift from the exploitation of natural enzymes and pathways to the design of recombinant cell factories comprising heterologous enzymes and/or synthetic, orthologous pathways for the synthesis of industrially relevant compounds. This review discusses recent developments and concepts applied in the frame of multistep whole-cell biocatalysis along with representative examples.
    Current Opinion in Biotechnology. 01/2014; 30:178–189.
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    ABSTRACT: Pseudomonas sp. VLB120 was isolated in Stuttgart, Germany, as a styrene degrading organism. The complete genome sequence includes genomic information of solvent tolerance mechanisms, metabolic pathways for various organic compounds, and the megaplasmid pSTY.
    Journal of Biotechnology 10/2013; · 3.18 Impact Factor
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    Chemie Ingenieur Technik 09/2013; 85(9). · 0.70 Impact Factor
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    ABSTRACT: During the last decades, biocatalysis became of increasing importance for chemical and pharmaceutical industries. Regarding regio- and stereospecificity, enzymes have shown to be superior compared to traditional chemical synthesis approaches, especially in C-O functional group chemistry. Catalysts established on a process level are diverse and can be classified along a functional continuum starting with single-step biotransformations using isolated enzymes or microbial strains towards fermentative processes with recombinant microorganisms containing artificial synthetic pathways. The complex organization of respective enzymes combined with aspects such as cofactor dependency and low stability in isolated form often favors the use of whole cells over that of isolated enzymes. Based on an inventory of the large spectrum of biocatalytic C-O functional group chemistry, this review focuses on highlighting the potentials, limitations, and solutions offered by the application of self-regenerating microbial cells as biocatalysts. Different cellular functionalities are discussed in the light of their (possible) contribution to catalyst efficiency. The combined achievements in the areas of protein, genetic, metabolic, and reaction engineering enable the development of whole-cell biocatalysts as powerful tools in organic synthesis.
    Chemical Society Reviews 03/2013; · 24.89 Impact Factor
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    ABSTRACT: Microbial physiology plays a crucial role in whole-cell biotransformations, especially for redox reactions depending on carbon and energy metabolism. In this study, regio- and enantioselective proline hydroxylation with recombinant Escherichia coli containing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter p4h mRNA levels indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase of resting cell activities above a certain threshold (depending on growth and assay temperature). Up to 5-fold higher activities were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that TCA cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of putA and putP encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered to be the most critical factors.
    Applied and Environmental Microbiology 03/2013; · 3.95 Impact Factor

Publication Stats

3k Citations
680.03 Total Impact Points


  • 2014
    • Helmholtz-Zentrum für Umweltforschung
      Leipzig, Saxony, Germany
    • Universität Stuttgart
      • Institute for Biochemical Engineering
      Stuttgart, Baden-Württemberg, Germany
  • 2005–2014
    • Technische Universität Dortmund
      • • Laboratory of Chemical Biotechnology (BT)
      • • Chair of Chemical Biology
      Dortmund, North Rhine-Westphalia, Germany
  • 2010–2012
    • Leibniz-Institut für Analytische Wissenschaften
      Dortmund, North Rhine-Westphalia, Germany
  • 2009
    • University of New South Wales
      • School of Biotechnology and Biomolecular Sciences (BABS)
      Kensington, New South Wales, Australia
    • TNO
      Delft, South Holland, Netherlands
  • 2008
    • Ewha Womans University
      Sŏul, Seoul, South Korea
  • 2003–2008
    • Delft University Of Technology
      • Department of Biotechnology
      Delft, South Holland, Netherlands
  • 1999–2006
    • Eawag: Das Wasserforschungs-Institut des ETH-Bereichs
      Duebendorf, Zurich, Switzerland
  • 1998–2006
    • ETH Zurich
      • Institute of Energy Technology
      Zürich, ZH, Switzerland
  • 2004
    • Hochschule für Technik Zürich
      Zürich, Zurich, Switzerland