Marcus Taupp

Publications of Marcus Taupp

• The art and design of functional metagenomic screens.

  Authors: Marcus Taupp, Keith Mewis, Steven J Hallam

  Current opinion in biotechnology. 03/2011; 22(3):465-72.

  This article summarizes general design principles for functional metagenomics. The focus is on Escherichia coli as an expression host, although alternative host-vector systems are discussed inThis article summarizes general design principles for functional metagenomics. The focus is on Escherichia coli as an expression host, although alternative host-vector systems are discussed in relation to optimizing gene recovery in activity-based screens. Examples of DNA isolation and enrichment approaches, library construction and phenotypic read-out are described with special emphasis on the use of high throughput technologies for rapid isolation of environmental clones encoding phenotypic traits of interest.

• A high throughput screen for biomining cellulase activity from metagenomic libraries.

  Authors: Keith Mewis, Marcus Taupp, Steven J Hallam

  Journal of visualized experiments : JoVE. 01/2011;

  Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degradeCellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

• Expression of recombinant proteins in the methylotrophic yeast Pichia pastoris.

  Authors: Maria Weidner, Marcus Taupp, Steven J Hallam

  Journal of visualized experiments : JoVE. 01/2010;

  Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system. As aProtein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system. As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1]. As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3]. Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4]. The vector used here (pPICZalphaA) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the alpha-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

• Large insert environmental genomic library production.

  Authors: Marcus Taupp, Sangwon Lee, Alyse Hawley, Jinshu Yang, Steven J Hallam

  Journal of visualized experiments : JoVE. 01/2009;

  The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approachesThe vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE http://www.epibio.com/item.asp?ID=385). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

• Direct trapping of formaldehyde formed via oxidative N-demethylation of N,N-dialkylarylamines by Bacillus megaterium using cysteamine derivatization.

  Authors: Marcus Taupp, Frank Heckel, Dag Harmsen, Peter Schreier

  Journal of microbiological methods. 12/2006; 67(2):357-62.

  Oxidative N-demethylation was measured by incubation experiments using Bacillus megaterium isolated from topsoil as a biocatalyst for the N-demethylation of the N,N-dialkylarylaminesOxidative N-demethylation was measured by incubation experiments using Bacillus megaterium isolated from topsoil as a biocatalyst for the N-demethylation of the N,N-dialkylarylamines N,N-dimethylaniline and N-ethyl-N-methylaniline. Formed formaldehyde, normally difficult to analyse in biological systems because of further metabolization, was successfully trapped and converted into thiazolidine by addition of cysteamine into the incubation media. Studies using N,N-di-(trideutero-methyl)-aniline and N-ethyl-N-(trideuteromethyl)-aniline as well as N,N-di-[methyl-(13)C]-aniline and N-ethyl-N-[methyl-(13)C]-aniline were performed to confirm that the N-demethylation proceeds via formaldehyde.

• Production of natural methyl anthranilate by microbial N-demethylation of N-methyl methyl anthranilate by the topsoil-isolated bacterium Bacillus megaterium.

  Authors: Marcus Taupp, Dag Harmsen, Frank Heckel, Peter Schreier

  Journal of agricultural and food chemistry. 12/2005; 53(24):9586-9.

  Bacillus megaterium, isolated in a screening process from topsoil, was used for N-demethylation of natural N-methyl methyl anthranilate to produce natural methyl anthranilate. Maximal productivity ofBacillus megaterium, isolated in a screening process from topsoil, was used for N-demethylation of natural N-methyl methyl anthranilate to produce natural methyl anthranilate. Maximal productivity of 70 mg/L/day was achieved under laboratory-scale conditions without further optimization. No byproducts were observed. Thus, production of "natural" methyl anthranilate using B. megaterium is a significant improvement over comparable already existing procedures.

• Opposite enantioselectivities of two phenotypically and genotypically similar strains of Pseudomonas frederiksbergensis in bacterial whole-cell sulfoxidation.

  Authors: Waldemar Adam, Frank Heckel, Chantu R Saha-Möller, Marcus Taupp, Jean-Marie Meyer, Peter Schreier

  Applied and environmental microbiology. 05/2005; 71(4):2199-202.

  Soil samples were screened to select microorganisms with the capability to oxidize organic sulfides into the corresponding sulfoxides with differential enantioselectivities. Several bacterial strainsSoil samples were screened to select microorganisms with the capability to oxidize organic sulfides into the corresponding sulfoxides with differential enantioselectivities. Several bacterial strains that preferentially produced the S-configured sulfoxide enantiomer were isolated. Surprisingly, one bacterial strain, genotypically and phenotypically characterized as Pseudomonas frederiksbergensis, selectively gave the R enantiomer. The finding that two apparently identical organisms displayed opposite enantioselectivities is novel for non-genetically modified organisms.

• Growth, virulence, and immunogenicity of Listeria monocytogenes aro mutants.

  Authors: Jochen Stritzker, Jozef Janda, Christoph Schoen, Marcus Taupp, Sabine Pilgrim, Ivaylo Gentschev, Peter Schreier, Gernot Geginat, Werner Goebel

  Infection and immunity. 11/2004; 72(10):5622-9.

  Mutants of Listeria monocytogenes with deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrierMutants of Listeria monocytogenes with deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA. aroA, aroB, and in particular aroE mutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of the aro mutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K2, suggesting that the aro mutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of the aro mutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and all aro mutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 10(4)-fold for the aroA, aroB, and aroA/B mutants and >10(5)-fold for the aroE mutant compared to the parent strain. Nevertheless, mice preimmunized with aro mutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 x 10(6) aroA, aroB, and aroA/B mutant bacteria were sufficient to obtain a protective T-cell response, while 5 x 10(8) aroE or aroA/E mutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating that Listeria strains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.

• Direct trapping of formaldehyde formed via oxidative N-demethylation of N,N-dialkylarylamines by Bacillus megaterium using cysteamine derivatization

  Authors: Marcus Taupp, Frank Heckel, Dag Harmsen, Peter Schreier

  Journal of Microbiological Methods.

  Oxidative N-demethylation was measured by incubation experiments using Bacillus megaterium isolated from topsoil as a biocatalyst for the N-demethylation of the N,N-dialkylarylaminesOxidative N-demethylation was measured by incubation experiments using Bacillus megaterium isolated from topsoil as a biocatalyst for the N-demethylation of the N,N-dialkylarylamines N,N-dimethylaniline and N-ethyl-N-methylaniline. Formed formaldehyde, normally difficult to analyse in biological systems because of further metabolization, was successfully trapped and converted into thiazolidine by addition of cysteamine into the incubation media. Studies using N,N-di-(trideutero-methyl)-aniline and N-ethyl-N-(trideuteromethyl)-aniline as well as N,N-di-[methyl-13C]-aniline and N-ethyl-N-[methyl-13C]-aniline were performed to confirm that the N-demethylation proceeds via formaldehyde.

• Biohydroxylation of N,N-dialkylarylamines by the isolated topsoil bacterium Bacillus megaterium

  Authors: Marcus Taupp, Frank Heckel, Dag Harmsen, Peter Schreier

  Enzyme and Microbial Technology.

  Topsoil microorganisms were screened for their acceptability of the standard substrate N,N-dimethylaniline in bacterial ‘whole-cell’ incubations. One bacterium converted N,N-dimethylaniline and wasTopsoil microorganisms were screened for their acceptability of the standard substrate N,N-dimethylaniline in bacterial ‘whole-cell’ incubations. One bacterium converted N,N-dimethylaniline and was identified as Bacillus megaterium by 16S rDNA analysis and DNA/DNA-hybridization. In contrast to the well-known C-hydroxylation by liver microsomes, leading to p-hydroxylation, B. megaterium formed o- and p-monohydroxylated products, i.e. N,N-dimethyl-2-aminophenol and N,N-dimethyl-4-aminophenol, both identified by gas chromatography–mass spectrometry (GC–MS) using synthesized reference compounds. The observed hydroxylation showed slight regioselectivity in favour of the o-hydroxylated product. Two further substrates, N,N-diethylaniline and N-ethyl-N-methylaniline, were also successfully biohydroxylated by B. megaterium with corresponding regioselectivity. Interestingly, aniline, known to be transformed easily by cytochrome P-450meg into p-aminophenol, was not accepted as substrate.

• Biotransformation von N-Alkyl- und N,N-Dialkylarylaminen durch Bacillus megaterium

  Authors: Marcus Taupp

  In der vorliegenden Arbeit wurden Studien zur bakteriellen Biotransformation von N Alkyl- sowie N,N Dialkylarylaminen mit dem Ziel der arylischen Hydroxy-lierung und N-Dealkylierung durchgeführt.In der vorliegenden Arbeit wurden Studien zur bakteriellen Biotransformation von N Alkyl- sowie N,N Dialkylarylaminen mit dem Ziel der arylischen Hydroxy-lierung und N-Dealkylierung durchgeführt. Bodenproben wurden einem Screening-Verfahren unterworfen, um selektiv nach Mikroorganismen zu suchen, die zu den genannten Biotransformationen fähig waren. In einem Screeningverfahren wurden unter Verwendung von N-Ethyl-N methyl-anilin als Standardsubstrat aus Boden-proben Mikroorganismen isoliert und auf ihre Fähigkeit zur Biotransformation überprüft. Einer der isolierten Stämme setzte das zugeführte Substrat effizient und reproduzierbar zu drei Hauptprodukten um. Deren Strukturaufklärung erfolgte mittels Gaschromatographie-Massenspektrometrie (GC-MS) sowie anhand ein-dimensionaler NMR-Experimente (1H-NMR und 13C-NMR). Identifiziert wurden zwei hydroxylierte Produkte, N Ethyl-N-methyl-2-aminophenol und N-Ethyl-N-methyl-4-aminophenol, ferner wurde N-Ethylanilin, das N Demethylierungsprodukt gebildet. Die Identitäten wurden durch synthetisierte Referenzsubstanzen bestätigt. Die phänotypische und genotypische Charakterisierung dieses Bodenisolates als Bacillus megaterium erfolgte mittels mikroskopischer und färbetechnischer Methoden sowie der 16S rDNA-Sequenzierung. Zur eindeutigen Zuordnung wurde eine DNA/DNA-Hybridisierung gegenüber dem Bacillus megaterium Type-strain (DSM 32, ATCC 14581t) durchgeführt. In einem erweiterten Substratscreening wurden die strukturellen Voraussetzungen zur Biotransformation von N-Alkyl- und N,N Dialkylarylaminen durch Bacillus megaterium ermittelt. Ausführlich sind Sub-stituenteneffekte in Bezug auf Hydroxylierung und N-Dealkylierung untersucht worden. Sperrige und räumlich große Substrate wie N,N,N´,N´-Tetramethyl-p,p´ benzidin als auch N,N,N´,N´-Tetramethyl-p-benzidin wurden nicht hydro-xyliert; allerdings fand bei beiden Substrate eine N-Demethylierung statt, wobei das Erstere stärker N demethyliert wurde. Der Effekt des Austausches von Stickstoff gegen Phosphor in einigen Substraten wurde ebenfalls untersucht. So kamen Dimethylphenylphosphin und Diethylphenylphosphin mit Bacillus megaterium zur Anwendung. Phosphor-haltige Substrate wurden von B. megaterium nicht umgesetzt. Durch Versuche mit verschiedenen Induktoren und Repressoren wurde die Cytochrom-P-450-Aktvität des isolierten Bacillus megaterium bei der Bio-transformation von N,N-Diethylanilin untersucht. Hierbei wurde gezeigt, dass durch bestimmte Barbiturate die arylische Hydroxylierung als auch die N Deethylierung gesteigert wurden. Repression der Hydroxylierungs- und N Dealkylierungsaktivität zeigte sich durch Metyrapon, n-Octylamin, Pyridin und Imidazol. Die von Bacillus megaterium durchgeführte N-Demethylierung von N,N Dimethylanilin sowie N-Ethyl-N-methylanilin wurde im Hinblick auf Formaldehydbildung untersucht. Dies erfolgte im Inkubationsmedium direkt in-situ, mittels Cysteamin-Zugabe in das Medium, mit Umsetzung zu Thiazolidin. Anhand von Inkubationsversuchen mit den stabil-isotopenmarkierten Substraten N,N-Di-(trideuteromethyl)-anilin und N-Ethyl-N (trideuteromethyl)-anilin sowie N,N-Di-[methyl-13C]-anilin und N-Ethyl-N [methyl-13C]-anilin wurde anhand der Massenspektren der gebildeten Thiazolidine eindeutig festgestellt, dass die Methylgruppe als Formaldehyd abgespalten wird. Das Potential von Bacillus megaterium zur N-Dealkylierung wurde zur mikrobiellen N Demethylierung von natürlichem N-Methyl-methyl-anthranilat genutzt. Dadurch wurde der natürliche Aromastoff Methylanthranilat gewonnen. Zur Optimierung der bakteriellen Biotransformation von N-Methyl-methyl-anthranilat zu Methylanthranilat wurden in Bezug auf Wachstumsmedium, Inkubationstemperatur, pH-Wert des Inkubationsmediums sowie Substrat-konzentration verschiedene Variationen durchgeführt. Die antimikrobiellen Eigenschaften von N-Methyl-methylanthranilat und Methylanthranilat gegenüber Bacillus megaterium wurden durch Hemmhofbildung sowie der Bestimmung der Inhibierungskonzentration im Flüssigmedium nachgewiesen. Sowohl Substrat als auch Produkt erwiesen sich in den unterschiedlichen Tests als antibakteriell. Im Anschluß an Versuche zur Immobilisierung, die zu geringeren Ausbeuten führten, erfolgten schließlich Umsetzungen im Bioreaktor. Hierbei ließen sich die bei den Schüttelkulturen erhaltenen Ergebnisse direkt proportional auf den Fermenter übertragen. The goal of this study was to conduct the microbial biotransformation of N-alkyl- and N,N dialkylarylamines. Main objective was the arylic hydroxylation and N-dealkylation of this class of substrates. By this, microorganisms were isolated from topsoil and a selective screening process was started to search for microorganisms capable of con-ducting the biotransformations mentioned above. In a screening process and by the use of N-ethyl-N methylaniline as substrate, microorganisms of soil samples were isolated and proven of their ability to biotransformate N-ethyl-N-methylaniline. One isolate showed three biotransformation products. Structure determination was done by gaschromatography-masspectrometry (GC-MS) and nuclear magnetic resonance experiments (1H-NMR and 13C-NMR). N-ethyl-N-methylaniline was biohydroxylated to N-ethyl-N-methylaniline-2-aminophenol and N ethyl-N-methyl-4-aminophenol, N demethylation of N-ethyl-N-methyl-aniline afforded N-ethylaniline. The soil isolate was characterized and identified phenotypical and genotypical by colour reactions and the standard 16S rDNA method being Bacillus megaterium. To complete the assignment of the isolate to the species Bacillus megaterium, DNA/DNA-hybridization analysis against the Bacillus megaterium type strain (DSM 32, ATCC 14581t) was conducted. In an extended substrate screening, structural properties for a successful bio-transformation of N-alkyl- and N,N-dialkylarylamines by Bacillus megaterium were investigated. Substituent influences were proven in a further substrate screening regarding hydroxylation as well as N-dealkylation. Bulky and large substrates like N,N,N´,N´-tetramethyl-p,p´-benzidine and N,N,N´,N´-tetramethyl-p-benzidine were not hydroxylated by the microorganisms. But, N-demethylation was observed whereas the first one was more accepted. Exchange of nitrogen against phosphorus by using dimethylphenylphosphine and diethylphenylphosphine as substrates finally stopped all biotransformation activity of Bacillus megaterium. Neither arylic hydroxylation nor P dealkylation occurred. Trials to influence the bacterial activity towards the substrate N,N-diethylaniline, elicitors and inhibitors were used to affect cytochrome-P-450. Certain barbiturates clearly increased arylic hydroxylation as well as N dealkylation. Repression of these metabolites was shown by metyrapone, pyridine, n-octylamine and imidazole. N-demethylation reaction of Bacillus megaterium was further investigated for a detection of formaldehyde. Formaldehyde, built after N-demethylation of N,N dimethylaniline or N ethyl-N-methylaniline, was caught by added cysteamine and derivatized to thiazolidine. Incubation of the stable isotope labelled substrates N,N-di-(trideuteromethyl)-aniline and N-ethyl-N-(trideuteromethyl)-aniline as well as N,N-di[methyl 13C]-aniline and N-ethyl-N-[methyl-13C]-aniline and the resulting mass spectral fragmentations of the build labelled thiazolidines clearly stated that formaldehyde comes from methylgroups. Observed N-dealkylation reactions of Bacillus megaterium were used to produce the food flavour methyl anthranilate by microbial N-demethylation of N methyl methyl anthranilate. Several variations of the standard conditions were proven for the optimization of the bacterial biotransformation from N-methyl methyl anthranilate to methyl anthranilate: incubation temperature, pH-value, substrate concentration and media. Antimicrobial activities of N-methyl methyl anthranilate and methyl anthranilate against Bacillus megaterium were tested by using the paper disc diffusion assay and determination of the minimum inhibitory concentration in liquid medium. Substrate as well as product exhibited antimicrobial activity in both tests. To improve the stability of Bacillus megaterium, bacterial cells were immobilized in sodium-alginate-globules resulting in very low yields of methyl anthranilate. Finally, as a summarization of all obtained methyl anthranilate results, an upscaling was done in the bioreactor in a direct proportional way.