Showing papers by "Lars M. Blank published in 2018"

Memote: A community-driven effort towards a standardized genome-scale metabolic model test suite

Abstract: Several studies have shown that neither the formal representation nor the functional requirements of genome-scale metabolic models (GEMs) are precisely defined. Without a consistent standard, comparability, reproducibility, and interoperability of models across groups and software tools cannot be guaranteed. Here, we present memote (https://github.com/opencobra/memote) an open-source software containing a community-maintained, standardized set of metabolic model tests. The tests cover a range of aspects from annotations to conceptual integrity and can be extended to include experimental datasets for automatic model validation. In addition to testing a model once, memote can be configured to do so automatically, i.e., while building a GEM. A comprehensive report displays the model9s performance parameters, which supports informed model development and facilitates error detection. Memote provides a measure for model quality that is consistent across reconstruction platforms and analysis software and simplifies collaboration within the community by establishing workflows for publicly hosted and version controlled models.

From beech wood to itaconic acid: case study on biorefinery process integration.

Abstract: Renewable raw materials in sustainable biorefinery processes pose new challenges to the manufacturing routes of platform chemicals. Beside the investigations of individual unit operations, the research on process chains, leading from plant biomass to the final products like lactic acid, succinic acid, and itaconic acid is increasing. This article presents a complete process chain from wooden biomass to the platform chemical itaconic acid. The process starts with the mechanical pretreatment of beech wood, which subsequently is subjected to chemo-catalytic biomass fractionation (OrganoCat) into three phases, which comprise cellulose pulp, aqueous hydrolyzed hemicellulose, and organic lignin solutions. Lignin is transferred to further chemical valorization. The aqueous phase containing oxalic acid as well as hemi-cellulosic sugars is treated by nanofiltration to recycle the acid catalyst back to the chemo-catalytic pretreatment and to concentrate the sugar hydrolysate. In a parallel step, the cellulose pulp is enzymatically hydrolyzed to yield glucose, which—together with the pentose-rich stream—can be used as a carbon source in the fermentation. The fermentation of the sugar fraction into itaconic acid can either be performed with the established fungi Aspergillus terreus or with Ustilago maydis. Both fermentation concepts were realized and evaluated. For purification, (in situ) filtration, (in situ) extraction, and crystallization were investigated. The presented comprehensive examination and discussion of the itaconate synthesis process—as a case study—demonstrates the impact of realistic process conditions on product yield, choice of whole cell catalyst, chemocatalysts and organic solvent system, operation mode, and, finally, the selection of a downstream concept.

Heterologous production of long-chain rhamnolipids from Burkholderia glumae in Pseudomonas putida-a step forward to tailor-made rhamnolipids.

Abstract: Rhamnolipids are biosurfactants consisting of rhamnose (Rha) molecules linked through a β-glycosidic bond to 3-hydroxyfatty acids with various chain lengths, and they have an enormous potential for various industrial applications. The best known native rhamnolipid producer is the human pathogen Pseudomonas aeruginosa, which produces short-chain rhamnolipids mainly consisting of a Rha-Rha-C10-C10 congener. Bacteria from the genus Burkholderia are also able to produce rhamnolipids, which are characterized by their long-chain 3-hydroxyfatty acids with a predominant Rha-Rha-C14-C14 congener. These long-chain rhamnolipids offer different physicochemical properties compared to their counterparts from P. aeruginosa making them very interesting to establish novel potential applications. However, widespread applications of rhamnolipids are still hampered by the pathogenicity of producer strains and-even more important-by the complexity of regulatory networks controlling rhamnolipid production, e.g., the so-called quorum sensing system. To overcome encountered challenges of the wild type, the responsible genes for rhamnolipid biosynthesis in Burkholderia glumae were heterologously expressed in the non-pathogenic Pseudomonas putida KT2440. Our results show that long-chain rhamnolipids from Burkholderia spec. can be produced in P. putida. Surprisingly, the heterologous expression of the genes rhlA and rhlB encoding an acyl- and a rhamnosyltransferase, respectively, resulted in the synthesis of two different mono-rhamnolipid species containing one or two 3-hydroxyfatty acid chains in equal amounts. Furthermore, mixed biosynthetic rhlAB operons with combined genes from different organisms were created to determine whether RhlA or RhlB is responsible to define the fatty acid chain lengths in rhamnolipids.

A breath of information: the volatilome

Abstract: Volatile organic compounds (VOCs) are small molecular mass substances, which exhibit low boiling points and high-vapour pressures. They are ubiquitous in nature and produced by almost any organism of all kingdoms of life. VOCs are involved in many inter- and intraspecies interactions ranging from antimicrobial or fungal effects to plant growth promotion and human taste perception of fermentation products. VOC profiles further reflect the metabolic or phenotypic state of the living organism that produces them. Hence, they can be exploited for non-invasive medicinal diagnoses or industrial fermentation control. Here, we introduce the reader to these diverse applications associated with the monitoring and analysis of VOC emissions. We also present our vision of real-time VOC analysis enabled by newly developed analytical techniques, which will further broaden the use of VOCs in even wider applications. Hence, we foresee a bright future for VOC research and its associated fields of applications.

Restoration of biofuel production levels and increased tolerance under ionic liquid stress is enabled by a mutation in the essential Escherichia coli gene cydC.

Abstract: Microbial production of chemicals from renewable carbon sources enables a sustainable route to many bioproducts. Sugar streams, such as those derived from biomass pretreated with ionic liquids (IL), provide efficiently derived and cost-competitive starting materials. A limitation to this approach is that residual ILs in the pretreated sugar source can be inhibitory to microbial growth and impair expression of the desired biosynthetic pathway. We utilized laboratory evolution to select Escherichia coli strains capable of robust growth in the presence of the IL, 1-ethyl-3-methyl-imidizolium acetate ([EMIM]OAc). Whole genome sequencing of the evolved strain identified a point mutation in an essential gene, cydC, which confers tolerance to two different classes of ILs at concentrations that are otherwise growth inhibitory. This mutation, cydC-D86G, fully restores the specific production of the bio-jet fuel candidate d-limonene, as well as the biogasoline and platform chemical isopentenol, in growth medium containing ILs. Similar amino acids at this position in cydC, such as cydC-D86V, also confer tolerance to [EMIM]OAc. We show that this [EMIM]OAc tolerance phenotype of cydC-D86G strains is independent of its wild-type function in activating the cytochrome bd-I respiratory complex. Using shotgun proteomics, we characterized the underlying differential cellular responses altered in this mutant. While wild-type E. coli cannot produce detectable amounts of either product in the presence of ILs at levels expected to be residual in sugars from pretreated biomass, the engineered cydC-D86G strains produce over 200 mg/L d-limonene and 350 mg/L isopentenol, which are among the highest reported titers in the presence of [EMIM]OAc. The optimized strains in this study produce high titers of two candidate biofuels and bioproducts under IL stress. Both sets of production strains surpass production titers from other IL tolerant mutants in the literature. Our application of laboratory evolution identified a gain of function mutation in an essential gene, which is unusual in comparison to other published IL tolerant mutants.

Integration of genome-scale metabolic networks into whole-body PBPK models shows phenotype-specific cases of drug-induced metabolic perturbation.

Abstract: Drug-induced perturbations of the endogenous metabolic network are a potential root cause of cellular toxicity. A mechanistic understanding of such unwanted side effects during drug therapy is therefore vital for patient safety. The comprehensive assessment of such drug-induced injuries requires the simultaneous consideration of both drug exposure at the whole-body and resulting biochemical responses at the cellular level. We here present a computational multi-scale workflow that combines whole-body physiologically based pharmacokinetic (PBPK) models and organ-specific genome-scale metabolic network (GSMN) models through shared reactions of the xenobiotic metabolism. The applicability of the proposed workflow is illustrated for isoniazid, a first-line antibacterial agent against Mycobacterium tuberculosis, which is known to cause idiosyncratic drug-induced liver injuries (DILI). We combined GSMN models of a human liver with N-acetyl transferase 2 (NAT2)-phenotype-specific PBPK models of isoniazid. The combined PBPK-GSMN models quantitatively describe isoniazid pharmacokinetics, as well as intracellular responses, and changes in the exometabolome in a human liver following isoniazid administration. Notably, intracellular and extracellular responses identified with the PBPK-GSMN models are in line with experimental and clinical findings. Moreover, the drug-induced metabolic perturbations are distributed and attenuated in the metabolic network in a phenotype-dependent manner. Our simulation results show that a simultaneous consideration of both drug pharmacokinetics at the whole-body and metabolism at the cellular level is mandatory to explain drug-induced injuries at the patient level. The proposed workflow extends our mechanistic understanding of the biochemistry underlying adverse events and may be used to prevent drug-induced injuries in the future.

Discovery and Evaluation of Biosynthetic Pathways for the Production of Five Methyl Ethyl Ketone Precursors.

Abstract: The limited supply of fossil fuels and the establishment of new environmental policies shifted research in industry and academia toward sustainable production of the second generation of biofuels, with methyl ethyl ketone (MEK) being one promising fuel candidate. MEK is a commercially valuable petrochemical with an extensive application as a solvent. However, as of today, a sustainable and economically viable production of MEK has not yet been achieved despite several attempts of introducing biosynthetic pathways in industrial microorganisms. We used BNICE.ch as a retrobiosynthesis tool to discover all novel pathways around MEK. Out of 1325 identified compounds connecting to MEK with one reaction step, we selected 3-oxopentanoate, but-3-en-2-one, but-1-en-2-olate, butylamine, and 2-hydroxy-2-methylbutanenitrile for further study. We reconstructed 3 679 610 novel biosynthetic pathways toward these 5 compounds. We then embedded these pathways into the genome-scale model of E. coli, and a set of 18 622 were found to be the most biologically feasible ones on the basis of thermodynamics and their yields. For each novel reaction in the viable pathways, we proposed the most similar KEGG reactions, with their gene and protein sequences, as candidates for either a direct experimental implementation or as a basis for enzyme engineering. Through pathway similarity analysis we classified the pathways and identified the enzymes and precursors that were indispensable for the production of the target molecules. These retrobiosynthesis studies demonstrate the potential of BNICE.ch for discovery, systematic evaluation, and analysis of novel pathways in synthetic biology and metabolic engineering studies.

A model-based assay design to reproduce in vivo patterns of acute drug-induced toxicity

Abstract: Lars Kuepfer1 · Olivia Clayton2 · Christoph Thiel1 · Henrik Cordes1 · Ramona Nudischer2 · Lars M. Blank1 · Vanessa Baier1 · Stephane Heymans3,4 · Florian Caiment5 · Adrian Roth2 · David A. Fluri6 · Jens M. Kelm6 · José Castell7 · Nathalie Selevsek8 · Ralph Schlapbach8 · Hector Keun9 · James Hynes10 · Ugis Sarkans11 · Hans Gmuender12 · Ralf Herwig13 · Steven Niederer14 · Johannes Schuchhardt15 · Matthew Segall16 · Jos Kleinjans5

Evolutionary freedom in the regulation of the conserved itaconate cluster by Ria1 in related Ustilaginaceae

Abstract: Itaconate is getting growing biotechnological significance, due to its use as a platform compound for the production of bio-based polymers, chemicals, and novel fuels. Currently, Aspergillus terreus is used for its industrial production. The Ustilaginaceae family of smut fungi, especially Ustilago maydis, has gained biotechnological interest, due to its ability to naturally produce this dicarboxylic acid. The unicellular, non-filamentous growth form makes these fungi promising alternative candidates for itaconate production. Itaconate production was also observed in other Ustilaginaceae species such as U. cynodontis, U. xerochloae, and U. vetiveriae. The investigated species and strains varied in a range of 0–8 g L−1 itaconate. The genes responsible for itaconate biosynthesis are not known for these strains and therefore not characterized to explain this variability. Itaconate production of 13 strains from 7 species known as itaconate producers among the family Ustilaginaceae were further characterized. The sequences of the gene cluster for itaconate synthesis were analyzed by a complete genome sequencing and comparison to the annotated itaconate cluster of U. maydis. Additionally, the phylogenetic relationship and inter-species transferability of the itaconate cluster transcription factor Ria1 was investigated in detail. Doing so, itaconate production could be activated or enhanced by overexpression of Ria1 originating from a related species, showing their narrow phylogenetic relatedness. Itaconate production by Ustilaginaceae species can be considerably increased by changing gene cluster regulation by overexpression of the Ria1 protein, thus contributing to the industrial application of these fungi for the biotechnological production of this valuable biomass derived chemical.

Genetic Optimization Algorithm for Metabolic Engineering Revisited.

Abstract: To date, several independent methods and algorithms exist for exploiting constraint-based stoichiometric models to find metabolic engineering strategies that optimize microbial production performance. Optimization procedures based on metaheuristics facilitate a straightforward adaption and expansion of engineering objectives, as well as fitness functions, while being particularly suited for solving problems of high complexity. With the increasing interest in multi-scale models and a need for solving advanced engineering problems, we strive to advance genetic algorithms, which stand out due to their intuitive optimization principles and the proven usefulness in this field of research. A drawback of genetic algorithms is that premature convergence to sub-optimal solutions easily occurs if the optimization parameters are not adapted to the specific problem. Here, we conducted comprehensive parameter sensitivity analyses to study their impact on finding optimal strain designs. We further demonstrate the capability of genetic algorithms to simultaneously handle (i) multiple, non-linear engineering objectives; (ii) the identification of gene target-sets according to logical gene-protein-reaction associations; (iii) minimization of the number of network perturbations; and (iv) the insertion of non-native reactions, while employing genome-scale metabolic models. This framework adds a level of sophistication in terms of strain design robustness, which is exemplarily tested on succinate overproduction in Escherichia coli.

Using quantitative systems pharmacology to evaluate the drug efficacy of COX-2 and 5-LOX inhibitors in therapeutic situations

Abstract: A quantitative analysis of dose–response relationships is essential in preclinical and clinical drug development in order to optimize drug efficacy and safety, respectively. However, there is a lack of quantitative understanding about the dynamics of pharmacological drug–target interactions in biological systems. In this study, a quantitative systems pharmacology (QSP) approach is applied to quantify the drug efficacy of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitors by coupling physiologically based pharmacokinetic models, at the whole-body level, with affected biological networks, at the cellular scale. Both COX-2 and 5-LOX are key enzymes in the production of inflammatory mediators and are known targets in the design of anti-inflammatory drugs. Drug efficacy is here evaluated for single and appropriate co-treatment of diclofenac, celecoxib, zileuton, and licofelone by quantitatively studying the reduction of prostaglandins and leukotrienes. The impact of rifampicin pre-treatment on prostaglandin formation is also investigated by considering pharmacokinetic drug interactions with diclofenac and celecoxib, finally suggesting optimized dose levels to compensate for the reduced drug action. Furthermore, a strong correlation was found between pain relief observed in patients as well as celecoxib- and diclofenac-induced decrease in prostaglandins after 6 h. The findings presented reveal insights about drug-induced modulation of cellular networks in a whole-body context, thereby describing complex pharmacokinetic/pharmacodynamic behavior of COX-2 and 5-LOX inhibitors in therapeutic situations. The results demonstrate the clinical benefit of using QSP to predict drug efficacy and, hence, encourage its use in future drug discovery and development programs. Drug efficacy is governed by both pharmacokinetics at the whole-body level and pharmacodynamic responses at the cellular scale. A team around Lars Kuepfer at Germany’s RWTH Aachen University applied a quantitative systems pharmacology (QSP) approach to assess the therapeutic potential for a set of drugs in the treatment of pain and inflammatory diseases. To this end, the authors integrated a cellular network model of arachidonic acid metabolism into physiologically based pharmacokinetic models to obtain a quantitative understanding about the dynamics of pharmacological drug–target interactions. The integrated multiscale model was used to address prototypical questions of drug development programs such as dose finding, drug–drug interactions and assessment of therapeutic efficacy. The results of the study demonstrate the benefits of using QSP in future drug development programs.

Determination of growth-coupling strategies and their underlying principles

Abstract: Metabolic coupling of product synthesis and microbial growth is a prominent approach for maximizing production performance. Growth-coupling (GC) also helps stabilizing target production and allows the selection of superior production strains by adaptive laboratory evolution. We have developed the computational tool gcOpt, which identifies knockout strategies leading to the best possible GC by maximizing the minimally guaranteed product yield. gcOpt implicitly favors solutions resulting in strict coupling of product synthesis to growth and metabolic activity while avoiding solutions inferring weak, conditional coupling. GC intervention strategies identified by gcOpt were examined for GC triggering principles under diverse conditions. Curtailing the metabolism to render product formation an essential carbon drain was identified as one major strategy generating strong coupling of metabolic activity and target synthesis. Impeding the balancing of cofactors and protons in the absence of target production was the underlying principle of all other strategies and further increased the GC strength of the aforementioned strategies. Thus, generating a dependency between supply of global metabolic cofactors and product synthesis appears to be advantageous in enforcing strong GC.

Improved microscale cultivation of Pichia pastoris for clonal screening

Abstract: Expanding the application of technical enzymes, e.g., in industry and agriculture, commands the acceleration and cost-reduction of bioprocess development. Microplates and shake flasks are massively employed during screenings and early phases of bioprocess development, although major drawbacks such as low oxygen transfer rates are well documented. In recent years, miniaturization and parallelization of stirred and shaken bioreactor concepts have led to the development of novel microbioreactor concepts. They combine high cultivation throughput with reproducibility and scalability, and represent promising tools for bioprocess development. Parallelized microplate cultivation of the eukaryotic protein production host Pichia pastoris was applied effectively to support miniaturized phenotyping of clonal libraries in batch as well as fed-batch mode. By tailoring a chemically defined growth medium, we show that growth conditions are scalable from microliter to 0.8 L lab-scale bioreactor batch cultivation with different carbon sources. Thus, the set-up allows for a rapid physiological comparison and preselection of promising clones based on online data and simple offline analytics. This is exemplified by screening a clonal library of P. pastoris constitutively expressing AppA phytase from Escherichia coli. The protocol was further modified to establish carbon-limited conditions by employing enzymatic substrate-release to achieve screening conditions relevant for later protein production processes in fed-batch mode. The comparison of clonal rankings under batch and fed-batch-like conditions emphasizes the necessity to perform screenings under process-relevant conditions. Increased biomass and product concentrations achieved after fed-batch microscale cultivation facilitates the selection of top producers. By reducing the demand to conduct laborious and cost-intensive lab-scale bioreactor cultivations during process development, this study will contribute to an accelerated development of protein production processes.

Physiologic and metabolic characterization of Saccharomyces cerevisiae reveals limitations in the synthesis of the triterpene squalene

Abstract: Heterologous synthesis of triterpenoids in Saccharomyces cerevisiae from its native metabolite squalene has been reported to offer an alternative to chemical synthesis and extraction from plant material if productivities can be increased.Here, we physiologically characterized a squalene overproducing S. cerevisiae CEN.PK strain to elucidate the effect of cultivation conditions on the production of this central triterpenoid precursor. The maximum achievable squalene concentration was substantially influenced by nutritional conditions, medium composition and cultivation mode. Batch growth on glucose resulted in minimal squalene accumulation, while squalene only significantly accumulated during ethanol consumption (up to 59 mg/gCDW), probably due to increased acetyl-CoA supply on this carbon source. Likewise, low squalene concentrations were observed in glucose-limited chemostat cultivations and improved up to 8-fold upon increasing the ethanol fraction in the feed. In those experiments, a constant, growth-rate-independent specific squalene accumulation rate (2.2 mg/gCDW/h) was recorded resulting in a maximal squalene loading of 30 mg/gCDW at low dilution rates with longer residence times. Coenzyme A availability was identified as possible bottleneck as increased vitamin concentrations, including the Coenzyme A precursor pantothenate, improved squalene titers in batch and chemostat cultivations. This analysis demonstrates that thorough physiologic characterization of production strains is valuable for the identification of bottlenecks already in early stages of strain development and for guiding further optimization efforts.

What happens in yeast during the Crabtree effect? : an investigation of S. cerevisiae’s volatile space

Abstract: of volatile metabolites that have been found by a literature investigation. Not only the volatile metabolites in question have been added, but also substances and reactions that connect them to metabolites already present in the model. In total, 225 metabolites and 219 reactions were added to the model. Furthermore, 12 metabolic reactions could be verified by physiological and enzyme assays of knockout mutants.

Mass spectrometric characterization of siderophores produced by Pseudomonas taiwanensis VLB120 assisted by stable isotope labeling of nitrogen source

Abstract: The structures of three previously unknown siderophores produced by the fluorescent, biotechnologically relevant Pseudomonas taiwanensis (P. taiwanensis) VLB120 bacteria were elucidated by means of hydrophilic interaction liquid chromatography (HILIC) hyphenated to high-resolution tandem mass spectrometry (HRMS/MS). They could be verified as iron(III)- and aluminum(III) complexes as well as the protonated molecules of the siderophores formed by in-source fragmentation. The siderophores were separated according to their different acyl side chains and additionally according their central ions. One of the siderophores was identified as pyoverdine with a malic acid (hydroxy succinic acid) amide side chain and a peptide moiety consisting of Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. The other analytes were assigned to an azotobactin with the identical peptide chain linked to the characteristic chromophoric unit and a pyoverdine with a variation in the amino acid sequence. Proline is directly linked to the pyoverdine chromophore instead of ornithine. Acidic and enzymatic hydrolyses were carried out to analyze the individual amino acids. Beside OHAsn, each amino acid of the peptide part was identified by HILIC-HRMS and comparison to authentic standards. Additionally, 15N-labeled cellular supernatants were analyzed by means of HRMS/MS. The results of the MS/MS experiments complemented by accurate mass data facilitated elucidation of the structures studied in this work and provided further confirmation of the three recently described pyoverdines of P. taiwanensis VLB120 (Baune et al. in Biometals 30:589-597, 2017. https://doi.org/10.1007/s10534-017-0029-7 ).

Multi-capillary Column Ion Mobility Spectrometry of Volatile Metabolites for Phenotyping of Microorganisms.

Abstract: Rational strain engineering requires solid testing of phenotypes including productivity and ideally contributes thereby directly to our understanding of the genotype-phenotype relationship. Actually, the test step of the strain engineering cycle becomes the limiting step, as ever advancing tools for generating genetic diversity exist. Here, we briefly define the challenge one faces in quantifying phenotypes and summarize existing analytical techniques that partially overcome this challenge. We argue that the evolution of volatile metabolites can be used as proxy for cellular metabolism. In the simplest case, the product of interest is a volatile (e.g., from bulk alcohols to special fragrances) that is directly quantified over time. But also nonvolatile products (e.g., from bulk long-chain fatty acids to natural products) require major flux rerouting that result potentially in altered volatile production. While alternative techniques for volatile determination exist, rather few can be envisaged for medium to high-throughput analysis required for phenotype testing. Here, we contribute a detailed protocol for an ion mobility spectrometry (IMS) analysis that allows volatile metabolite quantification down to the ppb range. The sensitivity can be exploited for small-scale fermentation monitoring. The insights shared might contribute to a more frequent use of IMS in biotechnology, while the experimental aspects are of general use for researchers interested in volatile monitoring.

Metabolic engineering of microbial hosts for the biosynthesis of high molecular weight hyaluronan

Abstract: N-acetylglucosamine, as ingredient in medical and cosmetic products rises steadily. Especially the niche of high molecular weight (MW) HA requires attention, as isolation from animal sources prevails here. The traditional HA production processes are neither efficient nor environmentally friendly, therefore, the present work aimed at establishing a microbial host for high MW hyaluronan production. By synthetic biology means, the use of the alternative carbon sources sucrose and N-acetylglucosamine, directly feeding into the precursor synthesis pathways, was aimed at.

A physiology-based model of bile acid metabolism shows altered tissue concentrations after drug administration and in specific genotype subgroups

Abstract: Drug-induced liver injuries (DILI) are an important issue in drug development and patient safety and often lead to termination of drug-development programs or late withdrawals of drugs. Since DILI events are hard to diagnose in preclinical settings, a need for alternative prediction methods such as computational modeling emerges. Impairment of bile acid (BA) metabolism, known as cholestasis, is a frequent form of DILI. Being rather a systemic then a single organ related disease, whole-body physiology-based modeling is a predestined approach for cholestasis modeling. The objectives of the presented study were 1) the development of a physiology-based model for human bile acid metabolism, 2) model validation and characterization for a virtual population, and 3) prediction and quantification of the effects of genetic predispositions and drug interaction on bile acid metabolism. The developed physiology-based bile acid (PBBA) model is based on the standard PBPK model of PKSim® and describes the bile acid circulation in a healthy reference individual. Active processes such as the hepatic synthesis, gallbladder emptying upon meal intake, transition through the gastrointestinal tract, reabsorption into the liver, distribution within the body, and excretion are included. The kinetics of active processes for the surrogate BA glycochenodeoxycholic acid were fitted to time-concentration profiles of blood BA levels reported in literature. The robustness of our PBBA model is underlined by the comparison of simulated plasma BA concentrations in a virtual population of 1,000 healthy individuals with reported data. In addition to plasma concentrations, the PBBA model allows simulations of BA exposure in relevant tissues like the liver and can therefore enhance the mechanistic understanding of cholestasis. This feature was used to analyse the reported increased risk of cholestatic DILI in Benign Recurrent Intrahepatic Cholestasis type 2 (BRIC2) patients. Simulations of the PBBA model suggest a higher susceptibility of BRIC2 patients towards cholestatic DILI due to BA accumulation in hepatocytes. Apart from these intrinsic effects, drug-interactions and their effect on the systemic bile acid metabolism were simulated by combining the PBBA model with a drug PBPK model of cyclosporine A (CsA). The results of which confirmed the reported higher risk of developing DILI as a consequence of CsA intake. Altogether, the presented model enhances our mechanistic understanding of cholestasis, allows the identification of drug-interactions leading to altered BA levels in blood and organs, and could be used to prevent clinical cases of cholestasis and enhance patient safety.

Malatproduktion aus Rohglycerin mit Ustilago

Abstract: The use of biodiesel derived crude glycerol for the production of value added chemicals has been discussed frequently. Current glycerol-based microbial production process often suffers from low rates, titers, and yields, hindering their industrial application. Here we report on the identification and optimization of Ustilago trichophora as promising novel production organism for malic acid from glycerol. All optimization steps presented avoid metabolic engineering resulting in a non-GMO organism.

Discovery and Evaluation of Biosynthetic Pathways for the Production of Five Methyl Ethyl Ketone Precursors

Abstract: The limited supply of fossil fuels and the establishment of new environmental policies shifted research in industry and academia towards sustainable production of the 2nd generation of biofuels, with Methyl Ethyl Ketone (MEK) being one promising fuel candidate. MEK is a commercially valuable petrochemical with an extensive application as a solvent. However, as of today, a sustainable and economically viable production of MEK has not yet been achieved despite several attempts of introducing biosynthetic pathways in industrial microorganisms. We used BNICE.ch to discover all novel pathways around MEK. Out of 1325 identified compounds connecting to MEK with one reaction step, we selected 3-oxopentanoate, but-3-en-2-one, but-1-en-2-olate, butylamine, and 2-hydroxy-2-methyl-butanenitrile for further study. We reconstructed 3679610 novel biosynthetic pathways towards these 5 compounds. We then embedded these pathways into the genome-scale model of E. coli, retaining a set of 18925 most biologically viable ones based on their thermodynamic feasibilities and yields. For each novel reaction in the viable pathways, we proposed the most similar KEGG reactions, with their gene and protein sequences, as candidates for either a direct experimental implementation or as basis for enzyme engineering. Through pathway similarity analysis we classified the pathways and identified the enzymes and precursors that were indispensable for the production of the target molecules. The developments from this study enhance the potential of BNICE.ch for discovery, systematic evaluation, and analysis of novel pathways in future synthetic biology and metabolic engineering studies.

Designer rhamnolipid production

Abstract: Till Tiso, iAMB Institute of Applied Microbiology, RWTH Aachen University, Aachen, Germany till.tiso@rwth-aachen.de Andrea Germer, iAMB Institute of Applied Microbiology, RWTH Aachen University, Aachen, Germany Conrad Müller, iAMB Institute of Applied Microbiology, RWTH Aachen University, Aachen, Germany Lars M. Blank, iAMB Institute of Applied Microbiology, RWTH Aachen University, Aachen, Germany