Martin Roth

Generation of larger numbers of separated microbial populations by cultivation in segmented-flow microdevices

The high speed production of fluid segments for the highly parallelized cultivation of monoclonal cell populations was carried out by the use of microchip segmentor modules. Aqueous fluid segments, embedded in a non-miscible carrier liquid, were produced with frequencies up to 30 s(-1) and showed a high homogeneity in size. This corresponds with the production of about 2.5 million samples per day. The segment volumes can be adapted between about 4 nl and 100 nl. The typical segment size for cultivation experiments is in the range between 40 nl and 80 nl. Nutrient medium can be applied instead of pure water. It is possible to aliquot a cell suspension in such a way that most of the aqueous fluid segments contain only one cell. In model experiments with four microbial species chip-produced aliquots of 60 nl, each containing one or a few cells, were incubated in Teflon capillary tubes. Rapid growth of the microcultures was observed. Cell densities were found to be as high as in conventional shake flask cultures.

Maintenance and genetic stability of vector plasmids pBR322 and pBR325 in Escherichia coli K12 strains grown in a chemostat

SummaryThe maintenance and genetic stability of the vector plasmids pBR322 and pBR325 in two genetically different Escherichia coli hosts were studied during chemostat cultivation with glucose and ammonium chloride limitation and at two different dilution rates. The plasmid pBR322 was stably maintained under all growth conditions tested. However pBR325 segregated from both hosts preferentially during glucose limitation and at low dilution rate. In addition to this general segregation process a separate loss of tetracycline resistance was observed. The remaining plasmid conferred resistance to ampicillin and chloramphenicol only, without any remarkable alteration of its molecular weight.Cultivation conditions in the chemostat were found that allowed the stable genetic inheritance of both plasmids in the hosts studied.

Polyketide‐Chain Branching by an Enzymatic Michael Addition

A new branch for polyketide synthases was discovered in the biosynthesis of the antimitotic rhizoxin complex in the endofungal bacterium Burkholderia rhizoxinica. Genetic engineering and the structural elucidation of pathway intermediates revealed that a complex polyketide chain is branched at the beta position by an unprecedented conjugate addition of an acetyl building block to an acryloyl precursor (see scheme).

Genetic stability of differentiated functions in Streptomyces hygroscopicus in relation to conditions of continuous culture.

The genetic stability of the capacity of an improved strain of Streptomyces hygroscopicus to produce the macrolide antibiotic turimycin was investigated during long-term continuous culture. Dilution rate, growth-limiting substrate and culture temperature were varied. Certain culture conditions resulted in the stable propagation of the inoculated turimycin-producing population. Other conditions led to segregation of the initial population. Turimycin non-producing phenotypes appeared, and in each case the simultaneous loss of ability to form aerial mycelium was observed. The non-differentiating clones were found to be stable, without any reversion to the parental phenotype, indicating that a loss of genetic information probably took place.

Production of polyhydroxybutyrate by Ralstonia eutropha from protein hydrolysates

Abstract Polyhydroxybutyrate (PHB) was produced by Ralstonia eutropha DSMu200911348 (formerly Alicaligenes eutrophus) in media containing 20–30u2009gu2009l−1 casein peptone or casamino acids as sole sources of nitrogen. In fermentations using media based on casein peptone, permanent growth up to a cell dry mass of 65u2009gu2009l−1 was observed. PHB accumulated in cells up to 60%–80% of dry weight. The lowest yields were found in media without any trace elements or with casamino acids added only. The residual cell dry masses were limited to 10–15u2009gu2009l−1 and did not contain PHB. The highest productivity amounted to 1.2u2009gu2009PHBu2009l−1u2009h−1. The mean molecular mass of the biopolymer was determined as 750u2009kDa. The proportion of polyhydroxyvalerate was less than 0.2% in PHB. The bioprocess was scaled up to a 300-l plant. During a fermentation time of 39u2009h the cells accumulated PHB to 78%u2009w/w. The productivity was 0.98u2009gu2009PHBu2009l−1u2009h1.

Differential expression of silent polyketide biosynthesis gene clusters in chemostat cultures of Aspergillus nidulans

The genome of the fungal model organism Aspergillus nidulans harbors nearly 30 polyketide synthase genes, yet the majority of these genes remain silent in the absence of particular stimuli. In this study, environmental conditions such as low specific microbial growth rate as well as nitrate, orthophosphate and glucose limitations were simulated under a continuous cultivation regime to induce the expression of silent polyketide synthase genes. In addition to offline and online bioprocess parameters, the physiological equilibrium was defined at the transcript level in terms of indicator gene expression. The different cultivation parameters resulted in a differential expression of two polyketide synthase genes coding for the biosynthesis of a variety of phenolic compounds, such as orsellinic acid, lecanoric acid, emodins, chrysophanol, shamixanthone, and sanghaspirodin. Further investigation of the metabolome revealed the formation of a novel prenylated benzophenone derivative designated as pre-shamixanthone. Our data indicate that employing chemostat fermentations in combination with genome mining, transcriptome analysis and metabolic profiling represents a valuable approach for triggering cryptic biosynthetic pathways.

Imaging Mass Spectrometry and Genome Mining Reveal Highly Antifungal Virulence Factor of Mushroom Soft Rot Pathogen

Caught in the act: imaging mass spectrometry of a button mushroom infected with the soft rot pathogen Janthinobacterium agaricidamnosum in conjunction with genome mining revealed jagaricin as a highly antifungal virulence factor that is not produced under standard cultivation conditions. The structure of jagaricin was rigorously elucidated by a combination of physicochemical analyses, chemical derivatization, and bioinformatics.

Biosynthesis of the Respiratory Toxin Bongkrekic Acid in the Pathogenic Bacterium Burkholderia gladioli

Bongkrekic acid (BA), an infamous respiratory toxin of the pathogenic bacterium Burkholderia gladioli, causes lethal intoxications when tempe bongkrek is produced with contaminated Rhizopus oligosporus cultures. Genome sequencing of B. gladioli pathovar cocovenenans unveiled the genetic basis for BA biosynthesis, and pointed to a homologous bon gene cluster in a B. gladioli strain from an infected rice plant. For functional genetics in B. gladioli λ Red recombination was established. Dissection of the modular type I polyketide synthase (a trans-AT PKS) provided insights into complex polyketide assembly. Isoprenoid-like β-branching events and a six-electron oxidation of a methyl group to a carboxylic acid give rise to the unique branched tricarboxylic fatty acid. The role of the cytochrome P450 monooxygenase, BonL, was proven by structural elucidation of deoxybongkrekic acid from a mutant.

Reproducible Biofilm Cultivation of Chemostat-Grown Escherichia coli and Investigation of Bacterial Adhesion on Biomaterials Using a Non-Constant-Depth Film Fermenter

Biomaterials-associated infections are primarily initiated by the adhesion of microorganisms on the biomaterial surfaces and subsequent biofilm formation. Understanding the fundamental microbial adhesion mechanisms and biofilm development is crucial for developing strategies to prevent such infections. Suitable in vitro systems for biofilm cultivation and bacterial adhesion at controllable, constant and reproducible conditions are indispensable. This study aimed (i) to modify the previously described constant-depth film fermenter for the reproducible cultivation of biofilms at non-depth-restricted, constant and low shear conditions and (ii) to use this system to elucidate bacterial adhesion kinetics on different biomaterials, focusing on biomaterials surface nanoroughness and hydrophobicity. Chemostat-grown Escherichia coli were used for biofilm cultivation on titanium oxide and investigating bacterial adhesion over time on titanium oxide, poly(styrene), poly(tetrafluoroethylene) and glass. Using chemostat-grown microbial cells (single-species continuous culture) minimized variations between the biofilms cultivated during different experimental runs. Bacterial adhesion on biomaterials comprised an initial lag-phase I followed by a fast adhesion phase II and a phase of saturation III. With increasing biomaterials surface nanoroughness and increasing hydrophobicity, adhesion rates increased during phases I and II. The influence of materials surface hydrophobicity seemed to exceed that of nanoroughness during the lag-phase I, whereas it was vice versa during adhesion phase II. This study introduces the non-constant-depth film fermenter in combination with a chemostat culture to allow for a controlled approach to reproducibly cultivate biofilms and to investigate bacterial adhesion kinetics at constant and low shear conditions. The findings will support developing and adequate testing of biomaterials surface modifications eventually preventing biomaterial-associated infections.

Two Induced Fungal Polyketide Pathways Converge into Antiproliferative Spiroanthrones

Filamentous fungi, such as the Aspergilli, are renowned for their medical, biotechnological and agricultural importance. In addition to synthetically valuable biotransformations by fungal enzymes, fungi are of pre-eminent interest to chemists and pharmacists because of their capability to produce a vast array of complex compounds. Indeed, many fungal natural products have found application in modern medicine or have served as leads for the development of new therapeutics. 3] Yet, the analysis of fungal genomes shows that fungi have the potential to produce a much broader range of secondary metabolites than evident from previous chemical analyses. During the last decade, the advent of elaborate molecular techniques has spurred efforts to investigate the actual biosynthetic potential of microorganisms, and ample evidence indicates that the majority of natural product biosynthetic genes is not expressed under standard laboratory culture conditions. 6] Sequencing and mining entire microbial genomes provided an exciting tool to identify and elucidate such cryptic biosynthetic pathways. Using Aspergillus nidulans as a model organism, considerable progress was made in activating silent genes to get access to this untapped reservoir of potentially useful metabolites. Further experiments with different fungal genera have shown the potential and versatility of novel concepts, such as the modulation of the epigenetic regulation of biosynthetic genes. The ability to produce diverse secondary metabolites probably represents the prerequisite for a microorganism to adapt to varying environmental conditions in the habitat, and the secreted metabolites might be seen as a chemical armamentarium to secure a niche. Therefore, it is important to understand the physiological conditions under which these compounds are produced. Early reports of fermentation processes to improve antibiotic production have taught that the choice of culture parameters is critical to the quantity, the type and the number of secondary metabolites produced. Thus, a systematic variation of fermentation conditions can help to exploit microbial chemical diversity. However, common scale-up processes to create sufficient biomass for compound isolation usually involve a batch or fed-batch fermentation of the organism with an increase in biomass and creation of high cell densities. Under these conditions, variation and control of growth parameters is hampered. For this reason, here we applied a different approach. Using a chemostat we grew the fungus in a continuous culture system, thus providing a defined and constant environmental milieu. The advantage of such a system over a biologically heterogeneous batch culture is that the microbial population grows at a constant rate and environmental factors, such as pH, nutrient concentration, oxygen supply and light can be varied and controlled. By limiting the rate of availability of a single nutrient, a fixed growth rate can be achieved and maintained for a longer time. The growth rate is determined by the dilution rate of the chemostat. To demonstrate the strength of this approach, in this study we report the discovery of two novel polyketide metabolites, sanghaspirodins A (1) and B (2), from the model fungus A. nidulans grown in a chemostat under nitrogen limitation. First, we established a continuous culture system for the growth of the fungus under defined physiological conditions. We initially focused on a limited supply of the nitrogen source. The influx of sterile medium was balanced by the efflux of spent medium as well as living fungal cells and cell debris, thus allowing growth at equilibrium (Figure 1 A). In total, 50 L of fungal culture were generated. Investigation of the culture biomass extract by HPLC-DAD-HRESI-MS and dereplication with natural product databases revealed the presence of two so far unknown metabolites (Figure 1 B) with identical UV spectra and the same molecular composition (m/z 511 [M H] , C29H20O9). Both compounds were isolated by a combination of different chromatographic techniques, and their structures were elucidated by 1D and 2D NMR analyses (Scheme 1 A). The number of carbon atoms as deduced from HRESI-MS was confirmed by C NMR spectroscopic measurements. DEPT135 and H NMR experiments revealed the presence of seven aromatic methine carbons and three methyl as well as four hydroxyl functional groups. The assignment of proton and carbon atoms was accomplished by HSQC and HMBC experiments and three partial structures, an anthrone moiety and two highly functionalised aromatic rings could be assembled as the basic [a] Dr. K. Scherlach, Prof. Dr. C. Hertweck Department of Biomolecular Chemistry Leibniz Institute for Natural Product Research and Infection Biology (HKI) Beutenbergstrasse 11a, 07745 Jena (Germany) E-mail : christian.hertweck@hki-jena.de [b] A. Sarkar, Dr. M. Roth, Dr. U. Horn Bio Pilot Plant, Leibniz Institute for Natural Product Research and Infection Biology Beutenbergstrasse 11a, 07745 Jena (Germany) [c] Dr. V. Schroeckh, Prof. Dr. A. A. Brakhage Department of Molecular and Applied Microbiology Leibniz Institute for Natural Product Research and Infection Biology Beutenbergstrasse 11a, 07745 Jena (Germany) [d] Dr. H.-M. Dahse Department of Infection Biology Leibniz Institute for Natural Product Research and Infection Biology Beutenbergstrasse 11a, 07745 Jena (Germany) [e] Prof. Dr. A. A. Brakhage, Prof. Dr. C. Hertweck Friedrich Schiller University, Jena (Germany) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201100132.