Andreas Schmid

Helmholtz-Zentrum für Umweltforschung, Leipzig, Saxony, Germany

Are you Andreas Schmid?

Claim your profile

Publications (202)821.53 Total impact

  • Babu Halan · Rohan Karande · Katja Buehler · Andreas Schmid

    No preview · Article · Feb 2016
  • Rohan Karande · Andreas Schmid · Katja Buehler
    [Show abstract] [Hide abstract]
    ABSTRACT: During the last decade, there was a rapid progress in integrating microfluidic reactors and biocatalytic reactions for various applications. The combination of miniaturized technologies and microfluidics allowed coupling of scale and time-dependent phenomena for bioprocess intensification. However, coupling microreactors and biocatalysis is highly complex, requiring an integrated approach addressing biocatalyst features, reaction kinetics, mass transfer and reactor engineering, as outlined in this review. Dimensionless numbers are discussed, which help identifying rate limiting steps and offer opportunities to enhance the overall reaction performance in solid-liquid biocatalytic reactions. This integrated concept is realized in a case study based on the biocatalytic conversion of styrene to (S)-styrene oxide using catalytic biofilms to demonstrate the pivotal role of this integrated approach for evolving the optimal (micro)-reactor configuration and design.
    No preview · Article · Jan 2016 · Organic Process Research & Development
  • [Show abstract] [Hide abstract]
    ABSTRACT: The microbial production of isoprenoids has recently developed into a prime example for successful bottom-up synthetic biology or top-down systems biology strategies. Respective fermentation processes typically rely on growing recombinant microorganisms. However, the fermentative production of isoprenoids has to compete with cellular maintenance and growth for carbon and energy. Non-growing but metabolically active E. coli cells were evaluated in this study as alternative biocatalyst configurations to reduce energy and carbon loss towards biomass formation. The use of non-growing cells in an optimized fermentation medium resulted in more than fivefold increased specific limonene yields on cell dry weight and glucose, as compared to the traditional growing-cell-approach. Initially, the stability of resting cells was limited. This instability was overcome via the optimization of the minimal fermentation medium enabling high and stable limonene production rates for up to 8 h and a high specific yield of ≥ 50 mg limonene per g cell dry weight. Omitting MgSO4 from the fermentation medium was very promising to prohibit growth and allow high productivities. Applying a MgSO4 -limitation also improved limonene formation by growing cells during non-exponential growth, involving a reduced biomass yield on glucose, a 4-fold increase in specific limonene formation yields on biomass, as compared to non-limited cultures. The control of microbial growth via the medium composition was identified as a key but yet underrated strategy for efficient isoprenoid production. This article is protected by copyright. All rights reserved.
    No preview · Article · Nov 2015 · Biotechnology and Bioengineering
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Pivotal challenges in industrial biotechnology are the identification and overcoming of cell-to-cell heterogeneity in microbial processes. While the development of subpopulations of isogenic cells in bioprocesses is well described (intra-population variability), a possible variability between genetically identical cultures growing under macroscopically identical conditions (clonal variability) is not. A high such clonal variability has been found for the recombinant expression of the styrene monooxygenase genes styAB from Pseudomonas taiwanensis VLB120 in solvent-tolerant Pseudomonas putida DOT-T1E using the alk-regulatory system from P. putida GPo1. In this study, the oxygenase subunit StyA fused to eGFP was used as readout tool to characterize the population structure in P. putida DOT-T1E regarding recombinant protein content. Flow cytometric analyses revealed that in individual cultures, at least two subpopulations with highly differing recombinant StyA-eGFP protein contents appeared (intra-population variability). Interestingly, subpopulation sizes varied from culture-to-culture correlating with the specific styrene epoxidation activity of cells derived from respective cultures (clonal variability). In addition, flow cytometric cell sorting coupled to plasmid copy number (PCN) determination revealed that detected clonal variations cannot be correlated to the PCN, but depend on the combination of the regulatory system and the host strain employed. This is, to the best of our knowledge, the first work reporting that intra-population variability (with differing protein contents in the presented case study) causes clonal variability of genetically identical cultures. Respective impacts on bioprocess reliability and performance and strategies to overcome respective reliability issues are discussed.
    Full-text · Article · Oct 2015 · Frontiers in Microbiology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Five mutants of Pseudomonas taiwanensis VLB120ΔCeGFP showed significant autoaggregation when growing on defined carbohydrates or gluconate, while they grew as suspended cells on complex medium and on organic acids like citrate and succinate. Surprisingly, the respective mutations affected very different genes, although all five strains exhibited the same behaviour of aggregate formation. To elucidate the mechanism of the aggregative behaviour, the microbial adhesion to hydrocarbons (MATH) assay and contact angle measurements were performed that pointed to an increased cell surface hydrophobicity. Moreover, investigations of the outer layer of the cell membrane revealed a reduced amount of O-specific polysaccharides in the lipopolysaccharide of the mutant cells. To determine the regulation of the aggregation, reverse transcription quantitative real-time PCR was performed and, irrespective of the mutation, the transcription of a gene encoding a putative phosphodiesterase, which is degrading the global second messenger cyclic diguanylate, was decreased or even deactivated in all mutants. In summary, it appears that the trophic autoaggregation was regulated via cyclic diguanylate and a link between the cellular cyclic diguanylate concentration and the lipopolysaccharide composition of P. taiwanensis VLB120ΔCeGFP is suggested.
    No preview · Article · Oct 2015 · Applied Microbiology and Biotechnology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: This data article refers to the report Δ9-Tetrahydrocannabinolicacid synthase (THCAS) production in Pichia pastoris enables chemical synthesis of cannabinoids (Lange et. al. 2015). THCAS was produced on a 2 L lab scale using recombinant Pichia pastoris KM71 KE1. Enrichment of THCAS as a technically pure enzyme was realized using dialysis and cationic exchange chromatography. nLC-ESI-MS/MS analysis identified THCAS in different fractions obtained by cationic exchange chromatography.
    Full-text · Article · Sep 2015
  • Source

    Full-text · Dataset · Aug 2015
  • Kerstin Lange · Andreas Schmid · Mattijs K Julsing
    [Show abstract] [Hide abstract]
    ABSTRACT: Δ(9)-tetrahydrocannabinol (THC) is of increasing interest as a pharmaceutical and bioactive compound. Chemical synthesis of THC uses a laborious procedure and does not satisfy the market demand. The implementation of biocatalysts for specific synthesis steps might be beneficial for making natural product availability independent from the plant. Δ(9)-Tetrahydrocannabinolicacid synthase (THCAS) from C. sativa L. catalyzes the cyclization of cannabigerolic acid (CBGA) to Δ(9)-tetrahydrocannabinolic acid (THCA), which is non-enzymatically decarboxylated to THC. We report the preparation of THCAS in amounts sufficient for the biocatalytic production of THC(A). Active THCAS was most efficiently obtained from Pichia pastoris. THCAS was produced on a 2 L bioreactor scale and the enzyme was isolated by single-step chromatography with a specific activity of 73 U g(-1)totalprotein. An organic / aqueous two-liquid phase setup for continuous substrate delivery facilitated in situ product removal. In addition, THCAS activity in aqueous environments lasted for only 20 min whereas the presence of hexane stabilized the activity over 3 h. In conclusion, production of THCAS in Pichia pastoris Mut(S) KM71 KE1, subsequent isolation, and its application in a two-liquid phase setup enables the synthesis of THCA on a mg scale. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · Jul 2015 · Journal of Biotechnology
  • Source
    Eleni Theodosiou · Oliver Frick · Bruno Bühler · Andreas Schmid
    [Show abstract] [Hide abstract]
    ABSTRACT: Understanding the metabolism of the microbial host is essential for the development and optimization of whole-cell based biocatalytic processes, as it dictates production efficiency. This is especially true for redox biocatalysis where metabolically active cells are employed because of the cofactor/cosubstrate regenerative capacity endogenous in the host. Recombinant Escherichia coli was used for overproducing proline-4-hydroxylase (P4H), a dioxygenase catalyzing the hydroxylation of free L-proline into trans-4-hydroxy-L-proline with a-ketoglutarate (a-KG) as cosubstrate. In this whole-cell biocatalyst, central carbon metabolism provides the required cosubstrate a-KG, coupling P4H biocatalytic performance directly to carbon metabolism and metabolic activity. By applying both experimental and computational biology tools, such as metabolic engineering and (13)C-metabolic flux analysis ((13)C-MFA), we investigated and quantitatively described the physiological, metabolic, and bioenergetic response of the whole-cell biocatalyst to the targeted bioconversion and identified possible metabolic bottlenecks for further rational pathway engineering. A proline degradation-deficient E. coli strain was constructed by deleting the putA gene encoding proline dehydrogenase. Whole-cell biotransformations with this mutant strain led not only to quantitative proline hydroxylation but also to a doubling of the specific trans-4-L-hydroxyproline (hyp) formation rate, compared to the wild type. Analysis of carbon flux through central metabolism of the mutant strain revealed that the increased a-KG demand for P4H activity did not enhance the a-KG generating flux, indicating a tightly regulated TCA cycle operation under the conditions studied. In the wild type strain, P4H synthesis and catalysis caused a reduction in biomass yield. Interestingly, the ΔputA strain additionally compensated the associated ATP and NADH loss by reducing maintenance energy demands at comparably low glucose uptake rates, instead of increasing the TCA activity. The putA knockout in recombinant E. coli BL21(DE3)(pLysS) was found to be promising for productive P4H catalysis not only in terms of biotransformation yield, but also regarding the rates for biotransformation and proline uptake and the yield of hyp on the energy source. The results indicate that, upon a putA knockout, the coupling of the TCA-cycle to proline hydroxylation via the cosubstrate a-KG becomes a key factor constraining and a target to further improve the efficiency of a-KG-dependent biotransformations.
    Preview · Article · Jul 2015 · Microbial Cell Factories
  • [Show abstract] [Hide abstract]
    ABSTRACT: The applications of biocatalysts in chemical industries are characterized by activity, selectivity and stability. One key strategy to achieve high biocatalytic activity is the identification of novel enzymes with kinetics optimized for organic synthesis by Nature. The isolation of novel cytochrome P450 monooxygenase genes from Acidovorax sp. CHX100 and their functional expression in recombinant Pseudomonas taiwanensis VLB120 enabled efficient oxidation of cyclohexane to cyclohexanol. Although initial resting cell activities of 20 U gCDW (-1) were achieved, the rapid decrease in catalytic activity due to the toxicity of cyclohexane prevented synthetic applications. Cyclohexane toxicity was reduced and cellular activities stabilized over the reaction time by delivering the toxic substrate through the vapor phase and by balancing the aqueous phase mass transfer with the cellular conversion rate. The potential of this novel CYP enzyme was exploited by transferring the shake flask reaction to an aqueous-air segmented flow biofilm membrane reactor for maximizing productivity. Cyclohexane was continuously delivered via the silicone membrane. This ensured lower reactant toxicity and continuous product formation at an average volumetric productivity of 0.4 g Ltube (-1) h(-1) for several days. This highlights the potential of combining a powerful catalyst with a beneficial reactor design to overcome critical issues of cyclohexane oxidation to cyclohexanol. It opens new opportunities for biocatalytic transformations of compounds which are toxic, volatile and have low solubility in water. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · Jun 2015 · Biotechnology and Bioengineering
  • Jan Volmer · Andreas Schmid · Bruno Bühler
    [Show abstract] [Hide abstract]
    ABSTRACT: Industrial bioprocess development is driven by profitability and eco-efficiency. It profits from an early stage definition of process and biocatalyst design objectives. Microbial bioprocess environments can be considered as synthetic technical microbial ecosystems. Natural systems follow Darwinian evolution principles aiming at survival and reproduction. Technical systems objectives are eco-efficiency, productivity, and profitable production. Deciphering technical microbial ecology reveals differences and similarities of natural and technical systems objectives, which are discussed in this review in view of biocatalyst and process design and engineering strategies. Strategies for handling opposing objectives of natural and technical systems and for exploiting and engineering natural properties of microorganisms for technical systems are reviewed based on examples. This illustrates the relevance of considering microbial ecology for bioprocess design and the potential for exploitation by synthetic biology strategies.
    No preview · Article · Jun 2015 · Current Opinion in Microbiology
  • Karsten Lang · Katja Buehler · Andreas Schmid
    [Show abstract] [Hide abstract]
    ABSTRACT: The chiral building block (S)-3-hydroxyisobutyric acid [(S)-3-HIBA] was produced either by conversion of isobutyric acid or directly from glucose utilizing cells of Pseudomonas taiwanensis VLB120 B83 T7 as catalysts. This strain carries a point mutation in the gene encoding 3-hydroxyisobutyrate dehydrogenase (mmsB), leading to its inactivation. The maximal specific activity in resting-cell biotransformations using isobutyric acid as substrate was 4.9±0.4cdw-1. Overexpression of the 2-ketoisovalerate pathway genes alsS, ilvC, and ilvD, and the introduction of kivd encoding 2-ketoacid decarboxylase resulted in the efficient fermentative synthesis of (S)-3-HIBA directly from glucose. Up to 22 mM (2.3 L-1) (S)-3-HIBA were produced at 3.7±0.3 gcdw-1 in repeated batch experiments without observable product degradation. Utilizing a biofilm reactor it was possible to continuously produce up to 6 mM (0.63 L-1) of (S)-3-hydroxyisobutyric acid with a volumetric productivity of 1.32 mmol(S)-3-HIBA-1L-1. Overall, the conversion of isobutyric acid to (S)-3-HIBA was found to be the rate-limiting step, leading to the accumulation of a mixture of (S)-3-hydroxyisobutyric acid and isobutyric acid. This study demonstrates for the first time the production of (S)-3-HIBA from renewable carbon in shake flasks and biofilm reactors and sets the stage for further optimizations towards the efficient production of 3-HIBA and its derivatives in continuous fermentations.
    No preview · Article · May 2015 · Advanced Synthesis & Catalysis
  • Christian David · Katja Bühler · Andreas Schmid
    [Show abstract] [Hide abstract]
    ABSTRACT: The application of segmented flow on a Synechocystis sp. PCC 6803 biofilm prevented excessive biomass formation and clogging by fundamentally changing the structure of the microbial community. It was possible to continuously operate a capillary microreactor for 5 weeks, before the experiment was actively terminated. The biofilm developed up to a thickness of 70-120 µm. Surprisingly, the biofilm stopped growing at this thickness and stayed constant without any detachment events occurring afterwards. The substrates CO2 and light were supplied in a counter-current fashion. Confocal microscopy revealed a throughout photosynthetically active biofilm, indicated by the red fluorescence of photo pigments. This control concept and biofilm reaction setup may enable continuous light driven synthesis of value added compounds in future.
    No preview · Article · May 2015 · Journal of Industrial Microbiology
  • Karolin Schmutzler · Andreas Schmid · Katja Buehler
    [Show abstract] [Hide abstract]
    ABSTRACT: For the investigation and comparison of microbial biofilms, a variety of analytical methods have been established, all focusing on different growth stages and application areas of biofilms. In this study, a novel quantitative assay for analysing biofilm maturation under the influence of continuous flow conditions was developed using the interesting biocatalyst Pseudomonas taiwanensis VLB120. In contrast to other tubular-based assay systems, this novel assay format delivers three readouts using a single setup in a total assay time of 40 h. It combines morphotype analysis of biofilm colonies with the direct quantification of biofilm biomass and pellicle formation on an air/liquid interphase. Applying the Tube-Assay, the impact of the second messenger cyclic diguanylate on biofilm formation of P. taiwanensis VLB120 was investigated. To this end, 41 deletions of genes encoding for protein homologues to diguanylate cyclase and phosphodiesterase were generated in the genome of P. taiwanensis VLB120. Subsequently, the biofilm formation of the resulting mutants was analysed using the Tube-Assay. In more than 60 % of the mutants, a significantly altered biofilm formation as compared to the parent strain was detected. Furthermore, the potential of the proposed Tube-Assay was validated by investigating the biofilms of several other bacterial species.
    No preview · Article · May 2015 · Applied Microbiology and Biotechnology
  • Diego Salamanca · Rohan Karande · Andreas Schmid · Daniel Dobslaw
    [Show abstract] [Hide abstract]
    ABSTRACT: Acidovorax sp. CHX100 has a remarkable ability for growth on short cycloalkanes (C5–C8) as a sole source of carbon and energy under aerobic conditions via an uncharacterized mechanism. Transposon mutagenesis of Acidovorax sp. CHX100 revealed a novel cytochrome P450 monooxygenase (CYP450chx) which catalyzed the transformation of cyclohexane to cyclohexanol. Primer walking methods categorized CYP450chx as cytochrome P450 class I taking into account its operon structure: monooxygenase, FAD oxidoreductase, and ferredoxin. CYP450chx was successfully cloned and expressed in Escherichia coli JM109. The activity of CYP450chx was demonstrated by means of the indole co-oxidation. Biotransformation capability of CYP450chx was confirmed through the catalysis of cycloalkanes (C5–C8) to their respective cyclic alcohols.
    No preview · Article · May 2015 · Applied Microbiology and Biotechnology
  • [Show abstract] [Hide abstract]
    ABSTRACT: The formation of stable emulsions in biphasic biotransformations catalyzed by microbial cells turned out to be a major hurdle for industrial implementation. Recently, a cost-effective and efficient downstream-processing approach, using supercritical carbon dioxide (scCO2 ) for both irreversible emulsion destabilization (enabling complete phase separation within minutes of emulsion treatment) and product purification via extraction has been proposed by Brandenbusch et al.(Biotechnology and Bioengineering 107:642-651, 2010). One of the key factors for a further development and scale-up of the approach is the understanding of the mechanism underlying scCO2 -assisted phase separation. A systematic approach was applied within this work to investigate the various factors influencing phase separation during scCO2 treatment (that is pressure, exposure of the cells to CO2 , and changes of cell surface properties). It was shown that cell toxification and cell disrupture are not responsible for emulsion destabilization. Proteins from the aqueous phase partially adsorb to cells present at the aqueous-organic interface, causing hydrophobic cell surface characteristics, and thus contribute to emulsion stabilization. By investigating the change in cell-surface hydrophobicity of these cells during CO2 treatment, it was found that a combination of catastrophic phase inversion and desorption of proteins from the cell surface is responsible for irreversible scCO2 mediated phase separation. These findings are essential for the definition of process windows for scCO2 -assisted phase separation in biphasic whole-cell biocatalysis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    No preview · Article · May 2015 · Biotechnology and Bioengineering
  • [Show abstract] [Hide abstract]
    ABSTRACT: Emulsion stability plays a crucial role for mass transfer and downstream processing in organic-aqueous bioprocesses based on whole microbial cells. In this study, emulsion stability dynamics and the factors determining them during two-liquid phase biotransformation were investigated for stereoselective styrene epoxidation catalyzed by recombinant Escherichia coli. Upon organic phase addition, emulsion stability rapidly increased correlating with a loss of solubilized protein from the aqueous cultivation broth and the emergence of a hydrophobic cell fraction associated with the organic-aqueous interface. A novel phase inversion-based method was developed to isolate and analyze cellular material from the interface. In cell-free experiments, a similar loss of aqueous protein did not correlate with high emulsion stability, indicating that the observed particle-based emulsions arise from a convergence of factors related to cell density, protein adsorption, and bioreactor conditions. During styrene epoxidation, emulsion destabilization occurred correlating with product-induced cell toxification. For biphasic whole-cell biotransformations, this study indicates that control of aqueous protein concentrations and selective toxification of cells enables emulsion destabilization and emphasizes that biological factors and related dynamics must be considered in the design and modeling of respective upstream and especially downstream processes.
    No preview · Article · Apr 2015 · Journal of Industrial Microbiology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Variability in whole-cell biocatalyst performance represents a critical aspect for stable and productive bioprocessing. In order to investigate whether and how oxygenase-catalyzed reactions are affected by such variability issues in solvent-tolerant Pseudomonas, different inducers, expression systems, and host strains were tested for the reproducibility of xylene and styrene monooxygenase catalyzed hydroxylation and epoxidation reactions, respectively. Significantly higher activity variations were found for biocatalysts based on solvent-tolerant Pseudomonas putida DOT-TIE and S12 compared with solvent-sensitive P. putida KT2440, Escherichia coli JM101, and solvent-tolerant Pseudomonas taiwanensis VLB120. Specific styrene epoxidation rates corresponded to cellular styrene monooxygenase contents. Detected variations in activity strictly depended on the type of regulatory system employed, being high with the alk- and low with the lac-system. These results show that the occurrence of clonal variability in recombinant gene expression in Pseudomonas depends on the combination of regulatory system and host strain, does not correlate with a general phenotype such as solvent tolerance, and must be evaluated case by case.
    No preview · Article · Apr 2015 · Journal of Industrial Microbiology
  • [Show abstract] [Hide abstract]
    ABSTRACT: The recent progress in sustainable chemistry and in synthetic biology increased the interest of chemical and pharmaceutical industries to implement microbial processes for chemical synthesis. However, most organisms used in biotechnological applications are not evolved by Nature for the production of hydrophobic, non-charged, volatile, or toxic compounds. In order to overcome this discrepancy, bioprocess design should consist of an integrated approach addressing pathway, cellular, reaction, and process engineering. Highlighting selected examples, we show that surprisingly often Nature provides conceptual solutions to enable chemical synthesis. Complemented by established methods from (bio)chemical and metabolic engineering, these concepts offer potential strategies yet to be explored and translated into innovative technical solutions enabling sustainable microbial production of non-natural chemicals. Copyright © 2015 Elsevier Ltd. All rights reserved.
    No preview · Article · Mar 2015 · Current Opinion in Biotechnology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM) on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4) comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m.)) deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s) for a single sample in contrast to 65 min by the LC-MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.
    Full-text · Article · Mar 2015 · EuPA Open Proteomics

Publication Stats

6k Citations
821.53 Total Impact Points


  • 2014-2015
    • Helmholtz-Zentrum für Umweltforschung
      Leipzig, Saxony, Germany
    • Universität Stuttgart
      • Institute for Biochemical Engineering
      Stuttgart, Baden-Württemberg, Germany
  • 2005-2015
    • Technische Universität Dortmund
      • • Laboratory of Chemical Biotechnology (BT)
      • • Chair of Chemical Biology
      Dortmund, North Rhine-Westphalia, Germany
  • 2013
    • Bielefeld University
      Bielefeld, North Rhine-Westphalia, Germany
  • 2010-2011
    • Leibniz-Institut für Analytische Wissenschaften
      Dortmund, North Rhine-Westphalia, Germany
  • 2008
    • Ewha Womans University
      Sŏul, Seoul, South Korea
  • 1999-2006
    • Eawag: Das Wasserforschungs-Institut des ETH-Bereichs
      Duebendorf, Zurich, Switzerland
  • 2004
    • Hochschule für Technik Zürich
      Zürich, Zurich, Switzerland
  • 1998-2000
    • ETH Zurich
      • Institute of Energy Technology
      Zürich, Zurich, Switzerland