DESIGN AND CONSTRUCTION OF BIOSENSING PLATFORMS FOR THE DETECTION OF BIOMARKERS

Ph.DDOCTOR OF PHILOSOPH

Identification and characterization of novel methyltransferases

So far 172 different types of nucleic acid modifications had been identified. However, for many RNA and DNA modifications the function(s) and the modifying enzymes (”writers”) are still not known. In my Ph.D. project, I focused on the identification of novel RNA and DNA methyltransferases. To identify novel enzymes, I systematically screened a collection of potential methyltransferases on the range of substrates in in vitro methyltransferase assay. I found four enzymes that exhibit robust methyltransferases activity towards RNA. Size exclusion chromatography in combination with labelling approaches and mass spectrometry allowed me to narrow down potential substrates and to identify the modified nucleosides for two candidate enzymes. I characterized METTL6 as a tRNA methyltransferases that specifically catalyzes methylation of cytosines at position 3 (m3C). By performing methyltransferase assays as well as sequence-specific purification of individual tRNAs, I identified the specific tRNA isoacceptors that are METTL6 targets and the precise position of the methylated C. RNA-seq and Ribosome profiling of KO mES cell lines revealed global changes in the transcriptome and translatome. In line with these changes, Mettl6 KO cells showed slower proliferation rates. Interestingly, METTL6 depletion resulted in slower hepatocellular tumor growth in in vitro models. In agreement with this, patients with high METTL6 expression levels have a reduced survival rate. I identified, METTL5 as a novel m6A RNA methyltransferase. Mass spectrometry analysis of RNA from Mettl5 KO mES cells revealed a decrease in m6A levels in 18S rRNA. By performing m6A-immunopreciptaion followed by sequencing in wt and Mettl5 KO cells, I mapped the transcript wide distribution of m6A in non-ribosomal RNAs. In addition to this, Mettl5 KO mES cells are less pluripotent and compromised in their ability to differentiate into neuronal precursors cells. In a complementary approach, I wanted to identify the interactome of METTL proteins. For this, I generated stable cells lines expressing tagged METTL family members, purified the complexes, and analysed interaction partners by mass spectrometry. The systematic identification of the complexes in which these potential enzymes act provides a useful resource for the epitranscriptomics community as it helps to understand METTL proteins function(s) and substrate specificities. Overall, in my Ph.D. project, I profiled the enzymatic activities and the interactome of 13 proteins of the METTL family. For two novel RNA methyltransferases, I characterized the substrates and the type of methylation they catalyse, profiled changes in the transcriptome and translatome, and characterized the phenotypes of mES cells upon KO of these two enzymes.Bisher wurden 172 verschiedene Arten von Nukleinsäuremodifikationen identifiziert. Für viele dieser RNA- und DNA-Modifikationen sind die Funktion (en) und die modifizierenden Enzyme ("Writer") jedoch noch nicht bekannt. In meinem Promotionsprojekt beschäftigte ich mich mit der Identifizierung neuartiger RNA- und DNA- Methyltransferasen. Um neue Enzyme zu identifizieren, habe ich systematisch eine Sammlung potenzieller Methyltransferasen in in-vitro-Methyltransferase-Assays auf verschiedenen Substraten untersucht. Ich habe vier Enzyme identifiziert, die robuste Methyltransferaseaktivität gegenüber RNA aufweisen. Größenausschlusschromatographie in Kombination mit Markierungsansätzen und Massenspektrometrie ermöglichten es mir, potenzielle Substrate einzugrenzen und die modifizierten Nukleoside für zwei positive Kandidaten zu identifizieren. Ich charakterisierte METTL6 als tRNA-Methyltransferase, die spezifisch m3C katalysiert. Durch Methyltransferase-Assays sowie sequenzspezifische Aufreinigung einzelner tRNAs identifizierte ich spezifische tRNA-Isoakzeptoren als METTL6-Tagets und die genaue Position des methylierten Cytosins. RNA-seq- und Ribosomen-Profiling von KO-mES-Zelllinien ergab globale Veränderungen im Transkriptom und Translatom. In Übereinstimmung mit diesen Änderungen zeigten Mettl6-KO-Zellen geringere Proliferationsraten. Interessanterweise führte METTL6-Depletion in in vitro-Modellen zu einem langsameren Wachstum hepatozellulärer Tumore. In Übereinstimmung damit haben Patienten mit hoher METTL6 Exoression eine verringerte Überlebensrate. Ich identifizierte METTL5 als eine neue m6A-RNA-Methyltransferase. Die massenspektrometrische Analyse von RNA aus Mettl5 KO mES-Zellen ergab eine Abnahme der Menge an m6A in 18S-rRNA. Durch das Durchführen von m6A- Immunpräzipationen, gefolgt von Sequenzierung in wt- und Mettl5 KO-Zellen, kartierte ich die transkriptweite Verteilung von m6A in nicht-ribosomalen RNAs. Diese METTL5-KO mES-Zellen verlieren Pluripotency und ihre Fähigkeit sich korrekt in neuronale Vorläuferzellen zu differenzieren. In einem komplementären Ansatz wollte ich das „Interaktom“ von METTL-Proteinen identifizieren. Zu diesem Zweck erzeugte ich stabile Zelllinien, die getagte METTL Protein exprimieren, reinigte die Komplexe und analysierte die Interaktionspartner durch Massenspektrometrie. Die systematische Identifizierung von Komplexen, in denen diese potenziellen Enzyme wirken, ist eine nützliche Ressource für das Epitranscriptomics Field, da sie zum Verständnis der Funktionen und Substratspezifitäten von METTL-Proteinen beitragen kann. Insgesamt habe ich in meiner Doktorarbeit die enzymatische Aktivität und das Interaktom von 13 Proteinen der METTL-Familie analysiert. Für zwei neue RNA- Methyltransferasen charakterisierte ich die Substrate und die Art der Methylierung, die sie katalysieren, profilierte Veränderungen im Transkriptom und Translatom und identifizierte die Phänotypen von mES-Zellen auf KO dieser beiden Enzyme

Sugars in early and late polyketide biosynthesis : Functional studies of rifL, rifK and rifM in rifamycin biosynthesis

The first section of this work comprises investigations concerning the initial steps in rifamycin biosynthesis. The amino sugar kanosamine was described to represent the most likely source of the nitrogen atom in the aminoshikimate pathway and is putatively synthesised by the catalytic activity of the enzymes RifL, RifK and RifM. Furthermore, in earlier studies kanosamine was even converted to kanosamine 6-phosphate by the rifamycin biosynthetic enzyme RifN. Therefore, evidence but no proof for the occurrence of kanosamine at an early stage in rifamycin biosynthesis was provided before the beginning of the present work. In this work functional studies of the gene products of rifL, rifK and rifM from the rifamycin producer Amycolatopsis mediterranei S699 were carried out using the plasmid pHGF7604 for the heterologous expression of the rifG-N genes in Streptomyces lividans TK24. Kanosamine was supposed to be synthesised by RifL, RifK and RifM during the RifL assay, employing a cell-free extract of Streptomyces lividans pHGF7604. These studies were based on former results concerning the RifL assay and aimed at the analytical detection of the putatively synthesised intermediate kanosamine en route to the known ansamycin precursor AHBA. Therefore, the RifL assay system was optimised, protein purification of cell-free extracts from Streptomyces lividans pHGF7604 was performed and analytical methods for kanosamine detection were developed within the scope of this study. The amino sugar was not detected in any RifL incubation sample, whereas concurrently no RT-PCR product of RifL could be verified. However, actual proof for an involvement of rifL, rifK and rifM in the postulated biosynthetic pathway of rifamycin could not be demonstrated under the terms of this study. The second section of this work is directed towards the characterisation of the gene cluster encoding the biosynthesis of the polycyclic xanthone IB-00208 in Streptomyces sp. GW2/5831. As polyketides represent an important source of nature-derived pharmaceuticals, their exploration provides promising opportunities for the discovery of new biogenic agents. Moreover, the acquirement of insights on the genetic level provides important information for genetic engineering that can lead to novel hybrid compounds with improved therapeutic values. In polyketide gene clusters “core” polyketide synthase (PKS) genes are encountered along with “post” PKS genes encoding modifying enzymes like oxygenases, methyl- and glycosyltransferases. In particular, glycosylation reactions that take place during post-PKS tailoring processes are relevant for genetic engineering. Especially genes encoding glycosyltransferases that catalyse (deoxy-) sugar attachments to the matured molecule are focussed on. The information obtained in this part of the work should provide a prerequisite for a prospective genetic engineering of the IB-00208 and griseorhodin A biosynthetic pathways. A glycosylated griseorhodin A molecule is expected to feature improved pharmacokinetic properties. As both compounds share the same polyaromatic core skeleton, it can be envisioned that griseorhodin A will be accepted as an aglycon-substrate by the glycosylating enzyme from the IB-00208 cluster. To isolate the IB-00208 cluster, a genomic library was established from DNA of Streptomyces sp. GW2/5831, and screening was performed employing ketosynthase (KS) primers. Sequencing of the positive cosmid clones and subsequent BLAST search allowed the deduction of a putative cluster fragment containing protein coding regions characteristic of this kind of PKS clusters, and genes possibly involved in the biosynthesis of IB-00208 were identified. The library was constructed using a shuttle vector that enabled the heterologous expression of the cosmid sequences in Streptomyces albus. This study sets the stage for further investigations of the IB- 00208 biosynthetic gene cluster from Streptomyces sp. GW2/5831. Both parts of the study are in the field of actinomycete genetics in drug development, representing different types of PKS systems. The genetics of streptomycetes is of particular importance, as it opens up many possibilities in the fields of biotechnology and pharmacy. Therefore many sequencing projects contribute to an enlarged knowledge on biosynthetic gene clusters of therapeutic agents that are of substantial interest

Metalloproteases involved in the Temozolomide (TMZ) resistance of U87-MG glioma cells

Glioblastoma Multiforme (GBM) ist der aggressivste hirneigene Tumor mit einer schlechten Überlebensprognose. Trotz Weiterentwicklung der multimodalen Behandlungsansätze, bestehend aus Tumorresektion und Radio- sowie Chemotherapie ist die Therapie des GBM bei einem durchschnittlichen Überleben von 2 Jahren nur bedingt erfolgreich. Als Standardchemotherapie kommt heutzutage das alkylierende Chemotherapeutikum Temozolomid (TMZ, Temodal®) zum Einsatz. Ein Grund für die ineffiziente Wirksamkeit von Chemo-und Radiotherapie ist die Ausbildung von Resistenzen in den Tumorzellen. Das Verständnis der Resistenzbildung auf molekularer Ebene ist daher sehr wichtig und könnte neue Ansätze zur Optimierung der Chemo- und Radiotherapie liefern. Metalloproteasen (MPs) sind in GBMs erhöht exprimiert und stets mit einer schlechten Prognose verbunden. In dieser Studie haben wir die Expression von MPs nach TMZ-Behandlung in U87-MG Glioblastom-Zellen systematisch untersucht. Eine TMZ-Behandlung von 5 Tagen induziert in U87-MG Zellen eine erhöhte Expression von ADAM8, MMP-1, MMP-9 and MMP-14 in den überlebenden Zellen. Um die Funktion der induzierten MPs zu untersuchen, wurde der Breitband MP-Inhibitor Batimastat (BB-94) in U87-MG Zellen in Verbindung mit TMZ eingesetzt. BB-94 führt zu einer erhöhten Sensitivität von U87-MG Zellen gegenüber dem TMZ-induzierten Zelltod und verringert das durch TMZ induzierte invasive Potenzial von U87-MG Zellen. Daraus folgt, dass die durch TMZ induzierten MPs sowohl zu einer Chemoresistenz als auch zu einer Rezidiv-Bildung beitragen können. Als potenzieller Signalweg der TMZ-vermittelten MP-Induktion in U87-MG Zellen wurde eine Zunahme der ERK1/2 Phosphorylierung unter TMZ-Behandlung nachgewiesen. Die Verwendung des ERK1/2 Inhibitors UO126 führte zu einer Abnahme der TMZ-induzierten Expression von ADAM8, MMP-1 and MMP-9, aber nicht von MMP-14, was darauf hindeutet, dass es alternative Signalwege bei der Induktion von MMP-14 gibt. PS/γ-Sekretase kombiniert mit MPs führt zu einer Prozessierung von Transmembran-Proteinen, durch die zahlreiche intrazelluläre Signalwege gesteuert werden. Die Inhibition von γ-Sekretase durch DAPT hat in unseren Analysen einen vergleichbaren Effekt wie BB-94 auf die Sensitivierung von U87-MG Zellen durch TMZ, was darauf schließen lässt, dass γ-Sekretase zusammen mit MPs zu einer Chemoresistenz von Glioblastom-Zellen führen kann. Zusammenfassend lässt sich sagen, dass die Induktion der Metalloproteasen MMP-1, MMP-9, MMP-14, and ADAM8 durch TMZ eine Chemoresistenz von Glioblastom-Zellen vermitteln kann und dass diese Metalloproteasen durch die Spaltung von Membranproteinen wie z.B. CD44, Met oder Integrinen an der „DNA Damage Response“ beteiligt sind oder die Proliferation und die Invasivität von Glioblastom-Zellen fördern können. Deshalb wäre eine kombinierte Therapie von TMZ mit Metalloprotease-Inhibitoren eine mögliche Therapie-Option, um die Effizienz der Standard-TMZ-Therapie zu verbessern und die Rezidivneigung zu verhindern.Glioblastoma multiforme (GBM) is the most malignant type of brain tumours bearing a grim prognosis with a median survival of 14.8 months. Despite therapeutic advances over the past decades, GBM treatment remains ineffective with temozolomide (TMZ) as a standard chemotherapeutic agent. Even using TMZ combined with surgical resection and radiotherapy, the 2-year survival for patients of high-grade gliomas remains very low. Understanding potential molecular mechanisms contributing to the resistance of glioma cells to TMZ is important for optimizing the existing and developing novel therapeutic strategies. Metalloproteases (MPs) have been found to be increased in GBM and are associated with the malignant phenotype. In our study, we have systematically examined the changes in the expression of MPs in U87-MG cells after TMZ treatment and found that treatment of U87-MG glioblastoma cells with TMZ for at least 5 days induces expression of metalloproteases ADAM8, MMP-1, MMP-9 and MMP-14 in surviving U87-MG cells. To analyse the function of these MPs, a broad-spectrum metalloprotease inhibitor batimastat (BB-94) was applied to U87-MG cells in conjunction with TMZ. BB-94 causes increased susceptibility of U87-MG cells to TMZ induced cell death and reduces the invasive potency of U87-MG cells, indicating that the induction of MPs by TMZ contributes to chemoresistance and recurrence of glioblastoma. As a potential mechanism of MP induction by TMZ we found that ERK1/2 phosphorylation in U87-MG cells is enhanced after TMZ treatment. Application of UO126, a specific ERK1/2 inhibitor, abrogates TMZ-induced expression of ADAM8, MMP-1 and MMP-9, but not of MMP-14, implicating that there are alternative pathways for the induction of MMP-14 by TMZ. PS/γ-secretase combined with MPs participates in trans-membrane processing of numerous proteins thereby regulating various intracellular pathways. Inhibition of γ-secretase using DAPT had a similar effect as BB-94 on sensitization of U87-MG cells to TMZ, suggesting that γ-secretase could function as an adjuvant peptidase to MPs to be involved in the chemoresistance of glioblastoma cells. Taken together, we conclude that the induction of particular MPs MMP-1, MMP-9, MMP-14, and ADAM8 by TMZ causes chemoresistance of glioblastoma cells, and these MPs confer chemoresistance via cleaving membrane-bound proteins, for example CD44, Met or integrins ß1 and/or alphav, which might be involved in DNA damage response (DDR) or in proliferation and invasiveness of tumour cells. Therefore, TMZ treatment combined with MP inhibitor appears as a feasible therapy option to optimize TMZ therapies and to prevent recurrent glioma formation

Effekten av stressfaktorer i matkjeden på Listeria monocytogenes

The work presented in this thesis was carried out at Section for Food Bacteriology, Unit Food Safety, Antimicrobial Resistance and Zoonoses, Research section for Food Safety and Animal Health at the Veterinary Institute and Department of Paraclinical Sciences at the Faculty of Veterinary Medicine NMBU in Oslo. The work was carried out between 2014 and 2019.Listeria monocytogenes is the causative agent of food-borne listeriosis, a disease often transmitted by contaminated Ready-To-Eat (RTE) food. Immunosuppressed individuals, fetuses, and the elderly are at higher risk of developing a lethal form of listeriosis. Despite preventive actions taken against L. monocytogenes contamination of food, human listeriosis is still a food safety challenge. L. monocytogenes is a robust ubiquitous soil bacterium that is highly resistant to several stressors in the food chain, from farm to the human host. It is therefore difficult to eradicate it from raw food material and food processing environments. However, avoiding contamination and subsequent inhibiting growth of the bacterium in the food are important and highly prioritized preventive measures taken by the food industry. L. monocytogenes’ ability to persist in the food environment and to cause severe disease makes it a costly foodborne pathogen to control both in the food industry and in the society. This PhD project has explored how L. monocytogenes copes with a selected set of stressors present in the food, in the environment and in the human host, focusing on the aim of increasing food safety.L. monocytogenes er årsaken til matbåren listeriose, en sykdom som ofte er forårsaket av spiseklar mat (Ready-To-Eat, RTE) som er forurenset med L. monocytogenes. Individer med nedsatt immunforsvar, fostre og eldre har høyere risiko for å få listeriose med dødelig utfall. Til tross for forebyggende tiltak mot L. monocytogenes i mat, er listeriose hos mennesker fortsatt en utfordring for mattryggheten. L. monocytogenes er en robust bakterie som finnes nesten overalt og som er svært resistent mot mange stressfaktorer i matkjeden. Den er derfor vanskelig å eliminere fra råvarer og fra matproduskjonsmiljøer. Til tross for dette er det høyt prioritert av matindustrien å iverksette forebyggende tiltak for å redusere forurensning med L. monocytogenes og påfølgende vekst av denne bakterien i matproduktene. L. monocytogenes evne til å persistere i miljøet og dens evne til å forårsake alvorlig sykdom gjør at den er en kostbar matbåren bakterie for både matindustrien og for samfunnet. Med mål om å styrke mattryggheten har dette PhD prosjektet undersøkt hvordan L. monocytogenes håndterer ulike stressfaktorer som finnes i mat, i miljøet og i mennesket som vertsorganisme.Veterinærinstitutte

Integrative assessment of low-dose gamma radiation effects on <i>Daphnia magna</i> reproduction: Toxicity pathway assembly and AOP development

High energy gamma radiation is potentially hazardous to organisms, including aquatic invertebrates. Although extensively studied in a number of invertebrate species, knowledge on effects induced by gamma radiation is to a large extent limited to the induction of oxidative stress and DNA damage at the molecular/cellular level, or survival, growth and reproduction at the organismal level. As the knowledge of causal relationships between effects occurring at different levels of biological organization is scarce, the ability to provide mechanistic explanation for observed adverse effects is limited, and thus development of Adverse Outcome Pathways (AOPs) and larger scale implementation into next generation hazard and risk predictions is restricted. The present study was therefore conducted to assess the effects of high-energy gamma radiation from cobalt-60 across multiple levels of biological organization (i.e., molecular, cellular, tissue, organ and individual) and characterize the major toxicity pathways leading to impaired reproduction in the model freshwater crustacean Daphnia magna (water flea). Following gamma exposure, a number of bioassays were integrated to measure relevant toxicological endpoints such as gene expression, reactive oxygen species (ROS), lipid peroxidation (LPO), neutral lipid storage, adenosine triphosphate (ATP) content, apoptosis, ovary histology and reproduction. A non-monotonic pattern was consistently observed across the levels of biological organization, albeit with some variation at the lower end of the dose-rate scale, indicating a complex response to radiation doses. By integrating results from different bioassays, a novel pathway network describing the key toxicity pathways involved in the reproductive effects of gamma radiation were proposed, such as DNA damage-oocyte apoptosis pathway, LPO-ATP depletion pathway, calcium influx-endocrine disruption pathway and DNA hypermethylation pathway. Three novel AOPs were proposed for oxidative stressor-mediated excessive ROS formation leading to reproductive effect, and thus introducing the world's first AOPs for non-chemical stressors in aquatic invertebrates.publishedVersio

Searching for biomarkers of non-alcoholic fatty liver disease and metabolic syndrome

151 p.El desarrollo de esta tesis doctoral ha aportado luz sobre los mecanismos que subyacen a la enfermedad del hígado graso no alcohólico (NAFLD por sus siglas en inglés), ya que los resultados han revelado la existencia de al menos 2 subtipos diferentes de pacientes al comparar (mediante cromatografía de líquidos acoplada a espectrometría de masas) el perfil metabolómico sérico de estos con el perfil metabolómico de dos modelos animales de esta enfermedad: ratones MAT1A-KO y 0.1MCD.Por otra parte, experimentos in vitro, in cellulo e in vivo han revelado parte del mecanismo de acción del Aramchol®, una molécula compuesta por un ácido biliar y un ácido graso saturado. El tratamiento con esta molécula en ratones 0.1MCD ha mostrado una clara reducción en la acumulación de grasa en el hígado, así como una disminución de la fibrosis y de un aumento en la capacidad antioxidante, todo ello disminuyendo los síntomas de esta enfermedad. Así mismo, el estudio clínico en humanos muestra el efecto del Aramchol en el metabolismo hepático de la glucosa, resultados que hemos complementado y validado en esta tesis con el modelo 0.1MCD.Por último, hemos caracterizado una cohorte de personas pertenecientes a población general, para determinar mediante Resonancia Magnética Nuclear (RMN) una huella metabólica en orina que nos permita distinguir pacientes con síndrome metabólico de personas sana

The role of the Fur protein for maintenance of iron homeostasis in the strictly anaerobic bacterium Clostridium acetobutylicum

Iron is an indispensable micronutrient for virtually all bacterial species. On the other hand, excess of iron could lead to formation of ROS, which could be toxic for the cell. Therefore, microorganisms have established a tight control on the intracellular iron content. The milestone of the bacterial iron-dependent response is the ferric uptake regulator (Fur) protein. In silico, biochemical and in vivo studies established the role of CAC1682 as a genuine iron-responsive regulator in the solvent-producing bacterium Clostridium acetobutylicum

Analysis of molecular interactions between yoghurt bacteria by an integrated genomics approach

The lactic acid bacteria (LAB) are a group of Gram-positive bacteria that ferment sugars such as lactose to produce mainly lactic acid. LAB are a group of industrially important microorganisms that are applied for the production of many fermented foods. These include foods produced with substrates from plant origin (e.g. sauerkraut and wine) and animal origin (e.g. fermented meats and dairy products such as yoghurt). The current market trends regarding sustainability and health-promoting foods demand more efficient and a more diverse range of fermentations. Most fermentations are carried out by multiple strains of different species. The interactions between consortium members are at the base of the performances of the individual microorganisms within a microbial ecosystem and therewith of the whole fermentation. These microbial interactions are often poorly understood. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are two LAB species that upon fermentation convert (bovine) milk into yoghurt. These two bacteria stimulate each other in growth and acid production. They produce exopolysaccharides (EPS), important for the texture of yoghurt, and characteristic flavor compounds such as acetaldehyde and diacetyl. However, the molecular basis of the mutualistic interactions between these two bacteria was poorly characterized. In this thesis research, a combination was used of screening, mixed culture transcription profiling, whole-genome metabolic modeling, experimental evolution and next-generation sequencing. This was done to unravel the molecular basis of the interactions between S. thermophilus and L. bulgaricus in milk. The results showed that interactions were primarily based on the exchange of metabolites (see Figure 1). Moreover, it was shown which genes or pathways were affected. Evidence was found that S. thermophilus provided L. bulgaricus with formic acid, folic acid (both involved in purine metabolism), long-chain fatty acids (by the action of lipolytic enzymes to break down milk fat) and CO2. The proteolysis by the exoprotease of L. bulgaricus, in turn, provided both species peptides, which are taken up by the cell and broken down into amino acids (AA) by intracellular peptidases. However, this probably did not yield a sufficient supply of branched-chain and sulfur AA, leading to a higher expression of the genes for biosynthesis of these AA in both species when grown in mixed culture. Moreover, EPS biosynthesis genes were induced in the mixed culture, leading to increased EPS production and a higher viscosity of the yoghurt. Figure 1. Schematic representation of the mutualistic interactions between S. thermophilus and L. bulgaricus in yoghurt. Solid arrows indicate interactions; dotted arrows indicate pathways that are affected by the interactions. Pathways that were for the first time shown to be regulated at the transcriptome level upon co-culture are indicated in bold. Pathways that were confirmed in our study to be regulated at the transcriptome level upon co-culture are underlined. EPS is hypothesized to promote the exchange of both bacteria. There was no evidence at the transcriptome level for the exchange of putrescine and ornithine. AA, amino acids; BCAA, branched-chain AA; EPS, exopolysaccharides; LCFA, long-chain fatty acids. A mixed culture genome-scale metabolic model confirmed that cross-feeding interactions between the yoghurt bacteria were based on purine and AA metabolism. Moreover, this model was used to show that the interactions provided a significant benefit to both bacteria, i.e. their biomass yield on lactose increased by around 50% in mixed culture. Experimental evolution revealed that it is possible to co-adapt a novel combination of strains of S. thermophilus and L. bulgaricus. It was shown that their mutual stimulation increased by optimizing their interactions by fine-tuning expression of pathways involved in the interactions. Furthermore, as little as ~1000 generations of co-culture was sufficient to transform the relatively slow growing mixed culture into one that showed similar performance as commercial starters with respect to key characteristics such as acidification rate and viscosity. Improved understanding of the described interactions that are at the base of the yoghurt fermentation provides us targets for the rational optimization of existing mixed culture fermentations and the rational development of industrially relevant mixed cultures, such as those containing probiotics. Moreover, the results are in particular interesting for the field of microbial ecology as they show how mutual nutritional dependencies evolve and structure the microbial composition of this ecosystem. <br/