Project acronym CHROMADAPT
Project The role of chromatin in the long-term adaptation of plants to abiotic stress
Researcher (PI) Isabel BÄURLE
Host Institution (HI) UNIVERSITAET POTSDAM
Call Details Consolidator Grant (CoG), LS9, ERC-2016-COG
Summary Abiotic stress is a major threat to global crop yields and this problem is likely to be exacerbated in the future. Therefore, it is very important to engineer crop plants with improved stress tolerance. A large body of research has focussed on the immediate stress responses. However, in nature stress is frequently chronic or recurring, suggesting that temporal dynamics are an important, but under-researched, component of plant stress responses. Indeed, plants can be primed by a stress exposure such that they respond more efficiently to the next stress incident. Such stress priming and memory may be particularly beneficial to plants due to their sessile life style. Typically, the memory of priming lasts for several days after the end of the stress. During the past few years, my group has initiated a molecular analysis of heat stress memory in Arabidopsis thaliana. Heat stress memory is associated with sustained gene induction and transcriptional memory and we have demonstrated that this involves lasting chromatin changes. The underlying molecular mechanisms, however, remain poorly understood. Here, I propose to combine mechanistic dissection of heat stress memory in A. thaliana with concomitant translation of the results into the temperate cereal crop barley. In particular, we will study the following questions: What is the role of chromatin during heat stress memory? How do the transcription factors involved mediate memory-specific outputs? How does nucleosome positioning affect heat stress memory? How do histone modifications during stress memory interact with transcription, chromatin and nuclear organization? Is heat stress memory conserved in temperate cereal species? Can we engineer plants with improved stress memory? Using existing tools and new methodologies, the proposed analyses will yield unprecedented insight into the long-term adaptation of plants to abiotic stress and open up approaches for breeding of stress-tolerant crops.
Summary
Abiotic stress is a major threat to global crop yields and this problem is likely to be exacerbated in the future. Therefore, it is very important to engineer crop plants with improved stress tolerance. A large body of research has focussed on the immediate stress responses. However, in nature stress is frequently chronic or recurring, suggesting that temporal dynamics are an important, but under-researched, component of plant stress responses. Indeed, plants can be primed by a stress exposure such that they respond more efficiently to the next stress incident. Such stress priming and memory may be particularly beneficial to plants due to their sessile life style. Typically, the memory of priming lasts for several days after the end of the stress. During the past few years, my group has initiated a molecular analysis of heat stress memory in Arabidopsis thaliana. Heat stress memory is associated with sustained gene induction and transcriptional memory and we have demonstrated that this involves lasting chromatin changes. The underlying molecular mechanisms, however, remain poorly understood. Here, I propose to combine mechanistic dissection of heat stress memory in A. thaliana with concomitant translation of the results into the temperate cereal crop barley. In particular, we will study the following questions: What is the role of chromatin during heat stress memory? How do the transcription factors involved mediate memory-specific outputs? How does nucleosome positioning affect heat stress memory? How do histone modifications during stress memory interact with transcription, chromatin and nuclear organization? Is heat stress memory conserved in temperate cereal species? Can we engineer plants with improved stress memory? Using existing tools and new methodologies, the proposed analyses will yield unprecedented insight into the long-term adaptation of plants to abiotic stress and open up approaches for breeding of stress-tolerant crops.
Max ERC Funding
1 998 525 €
Duration
Start date: 2017-05-01, End date: 2022-04-30
Project acronym conVIRgens
Project De- and reconstructing virulence strategies of fungal plant pathogens
Researcher (PI) Gunther DOEHLEMANN
Host Institution (HI) UNIVERSITAET ZU KOELN
Call Details Consolidator Grant (CoG), LS9, ERC-2017-COG
Summary Fungal pathogens are enormous threats to plants, causing tremendous losses in worldwide crop production. Mechanistic understanding of fungal virulence is crucial to developing novel plant protection strategies in sustainable agriculture.
Biotrophic pathogens colonize living plant tissue and reprogram their hosts to stimulate proliferation and development of infection structures. To promote infection, fungal pathogens secrete sets of virulence proteins termed “effectors” in a spatiotemporal program. Many economically relevant biotrophs like rusts and powdery mildew fungi are obligate pathogens. These organisms cannot be grown in culture and are not amenable to reverse genetics, which is a severe constraint for current research. In contrast, the biotrophic smut fungi have a haploid yeast stage, which allows simple cultivation and genetic modification. The causal agent of corn smut disease, Ustilago maydis, is one of the best-established model organisms for fungal genetics.
This project aims to utilize the excellent genetic accessibility of U. maydis to approach a previously impossible, pioneering enterprise: the synthetic reconstruction of eukaryotic plant pathogens. In a first step, fungal virulence will be deconstructed by consecutive deletion of the U. maydis effector repertoire to generate disarmed mutants. These strains will serve as chassis for subsequent reconstruction of fungal pathogenicity from different sources. A combination of transcriptomics and comparative genomics will help to define synthetic effector modules to reconstruct virulence in the chassis strains.
Deconstruction of U. maydis virulence will identify a complete arsenal of fungal virulence factors. Reconstruction of virulence will show how effector modules determine fungal virulence, including those of the previously not accessible obligate biotrophs. conVIRgens will thereby provide fundamentally new insights and novel functional tools towards the understanding of microbial virulence.
Summary
Fungal pathogens are enormous threats to plants, causing tremendous losses in worldwide crop production. Mechanistic understanding of fungal virulence is crucial to developing novel plant protection strategies in sustainable agriculture.
Biotrophic pathogens colonize living plant tissue and reprogram their hosts to stimulate proliferation and development of infection structures. To promote infection, fungal pathogens secrete sets of virulence proteins termed “effectors” in a spatiotemporal program. Many economically relevant biotrophs like rusts and powdery mildew fungi are obligate pathogens. These organisms cannot be grown in culture and are not amenable to reverse genetics, which is a severe constraint for current research. In contrast, the biotrophic smut fungi have a haploid yeast stage, which allows simple cultivation and genetic modification. The causal agent of corn smut disease, Ustilago maydis, is one of the best-established model organisms for fungal genetics.
This project aims to utilize the excellent genetic accessibility of U. maydis to approach a previously impossible, pioneering enterprise: the synthetic reconstruction of eukaryotic plant pathogens. In a first step, fungal virulence will be deconstructed by consecutive deletion of the U. maydis effector repertoire to generate disarmed mutants. These strains will serve as chassis for subsequent reconstruction of fungal pathogenicity from different sources. A combination of transcriptomics and comparative genomics will help to define synthetic effector modules to reconstruct virulence in the chassis strains.
Deconstruction of U. maydis virulence will identify a complete arsenal of fungal virulence factors. Reconstruction of virulence will show how effector modules determine fungal virulence, including those of the previously not accessible obligate biotrophs. conVIRgens will thereby provide fundamentally new insights and novel functional tools towards the understanding of microbial virulence.
Max ERC Funding
1 922 000 €
Duration
Start date: 2018-06-01, End date: 2023-05-31
Project acronym DeCoCt
Project Knowledge based design of complex synthetic microbial communities for plant protection
Researcher (PI) Eric Kemen
Host Institution (HI) EBERHARD KARLS UNIVERSITAET TUEBINGEN
Call Details Consolidator Grant (CoG), LS9, ERC-2018-COG
Summary "Complex microbial communities (""microbiota"") that populate surfaces of higher organisms critically impact health of their hosts: They contribute to vital functions such as host fitness, nutrient acquisition, stress tolerance and pathogen resistance but are, at the same time, reservoirs for facultative pathogens or can promote pathogenesis. How and why communities shift from a beneficial to a detrimental state is largely unknown and we are far from utilizing identified mechanisms.
In order to cure detrimental microbiota, that were damaged or reverted through stress factors including previous diseases, decoding the complex processes governing microbiota dynamics is a key challenge. To develop durable probiotics, communal stability or the ability of a community to return to a steady state following perturbation is a key factor.
Our lab has broad expertise in studying microbial communities through lab experiments and analyzing factors that shape the microbiota of Arabidopsis thaliana plants under natural conditions and common garden experiments. We have discovered a hierarchical order in microbial community networks with hub microbes as key elements. A recent breakthrough was the discovery of microbial taxa that persist throughout the life of A. thaliana plants and their importance in network stability.
In this project we will use our expertise to identify key stability factors and drivers of communal dynamics to reconstitute synthetic communities. How to seed microbial communities that develop into functional probiotics is a key challenge. We will use knowledge based assembly of complex communities to seeds protective microbiota. We will challenge those through pathogens and abiotic factors to refine and test the predictive power of our analyses. Therefore, DeCoCt represents a highly innovative approach that holds the potential to gain novel insights beyond the current scope of microbiota and probiotics research."
Summary
"Complex microbial communities (""microbiota"") that populate surfaces of higher organisms critically impact health of their hosts: They contribute to vital functions such as host fitness, nutrient acquisition, stress tolerance and pathogen resistance but are, at the same time, reservoirs for facultative pathogens or can promote pathogenesis. How and why communities shift from a beneficial to a detrimental state is largely unknown and we are far from utilizing identified mechanisms.
In order to cure detrimental microbiota, that were damaged or reverted through stress factors including previous diseases, decoding the complex processes governing microbiota dynamics is a key challenge. To develop durable probiotics, communal stability or the ability of a community to return to a steady state following perturbation is a key factor.
Our lab has broad expertise in studying microbial communities through lab experiments and analyzing factors that shape the microbiota of Arabidopsis thaliana plants under natural conditions and common garden experiments. We have discovered a hierarchical order in microbial community networks with hub microbes as key elements. A recent breakthrough was the discovery of microbial taxa that persist throughout the life of A. thaliana plants and their importance in network stability.
In this project we will use our expertise to identify key stability factors and drivers of communal dynamics to reconstitute synthetic communities. How to seed microbial communities that develop into functional probiotics is a key challenge. We will use knowledge based assembly of complex communities to seeds protective microbiota. We will challenge those through pathogens and abiotic factors to refine and test the predictive power of our analyses. Therefore, DeCoCt represents a highly innovative approach that holds the potential to gain novel insights beyond the current scope of microbiota and probiotics research."
Max ERC Funding
1 925 500 €
Duration
Start date: 2019-03-01, End date: 2024-02-29
Project acronym LUSH SPIKE
Project Genetic and Molecular Determinants of Spikelet Survival in Cereal Crops
Researcher (PI) Thorsten Schnurbusch
Host Institution (HI) LEIBNIZ - INSTITUT FUER PFLANZENGENETIK UND KULTURPFLANZENFORSCHUNG
Call Details Consolidator Grant (CoG), LS9, ERC-2015-CoG
Summary Meeting the forecasted world demand for food remains a crucial challenge for plant scientists in this century.
One promising avenue for improving grain yield of cereal crops, including wheat and barley, involves reducing spikelet mortality. Spikelets, the grain-bearing units of cereal spikes, usually form in excess and subsequently abort during development; increased spikelet survival is linked to increased numbers of grains per spike. Therefore, reducing spikelet mortality is an intriguing approach to improve grain yield.
In barley, the number of spikelets per spike at the awn primordium (AP) stage represents the maximum yield potential per spike. After the AP stage, significant spikelet mortality results in fewer grains per spike. Our previous results clearly indicated that spikelet survival in barley is highly genetically controlled (broad-sense heritability >0.80) and that the period from AP to tipping represents the most critical pre-anthesis phase related to spikelet reduction and grain yield per spike. However, the underlying genetic and molecular
determinants of spikelet survival remain to be discovered. I therefore propose this ambitious research program with an emphasis on using available genetic resources.
Our specific aims during the LUSH SPIKE project are to: (i) discover quantitative trait loci (QTL) for spikelet survival and grain number per spike and validate these QTL in bi-parental doubled-haploid mapping populations, (ii) isolate and functionally characterize Mendelized QTL using a map-based approach, (iii) reveal gene regulatory networks determining spikelet survival during the critical spike growth period from AP to heading, and (iv) elucidate spatio-temporal patterns of metabolite and phytohormone distributions in spike and spikelet sections during the critical growth period, using mass spectrometric imaging.
The results we obtain will advance our understanding of how to improve yields of cereal crops.
Summary
Meeting the forecasted world demand for food remains a crucial challenge for plant scientists in this century.
One promising avenue for improving grain yield of cereal crops, including wheat and barley, involves reducing spikelet mortality. Spikelets, the grain-bearing units of cereal spikes, usually form in excess and subsequently abort during development; increased spikelet survival is linked to increased numbers of grains per spike. Therefore, reducing spikelet mortality is an intriguing approach to improve grain yield.
In barley, the number of spikelets per spike at the awn primordium (AP) stage represents the maximum yield potential per spike. After the AP stage, significant spikelet mortality results in fewer grains per spike. Our previous results clearly indicated that spikelet survival in barley is highly genetically controlled (broad-sense heritability >0.80) and that the period from AP to tipping represents the most critical pre-anthesis phase related to spikelet reduction and grain yield per spike. However, the underlying genetic and molecular
determinants of spikelet survival remain to be discovered. I therefore propose this ambitious research program with an emphasis on using available genetic resources.
Our specific aims during the LUSH SPIKE project are to: (i) discover quantitative trait loci (QTL) for spikelet survival and grain number per spike and validate these QTL in bi-parental doubled-haploid mapping populations, (ii) isolate and functionally characterize Mendelized QTL using a map-based approach, (iii) reveal gene regulatory networks determining spikelet survival during the critical spike growth period from AP to heading, and (iv) elucidate spatio-temporal patterns of metabolite and phytohormone distributions in spike and spikelet sections during the critical growth period, using mass spectrometric imaging.
The results we obtain will advance our understanding of how to improve yields of cereal crops.
Max ERC Funding
2 000 000 €
Duration
Start date: 2016-07-01, End date: 2021-06-30
Project acronym OptoRibo
Project Optogenetic control of cellular behaviour by allosteric ribonucleic acid assemblies
Researcher (PI) Günter Mayer
Host Institution (HI) RHEINISCHE FRIEDRICH-WILHELMS-UNIVERSITAT BONN
Call Details Consolidator Grant (CoG), LS9, ERC-2013-CoG
Summary Light-sensitive channel proteins have attracted much attention as functional molecules, as they possess unique conformations and functions depending on their respective irradiation states. Embedding these proteins in heterogeneous cellular frameworks enabled to gain light-control of cellular and in particular neuronal behaviour. However to date, optogenetic solutions that address endogenous, intracellular biomolecules in a universal fashion remain elusive. The project will apply design and selection strategies aiming at nucleic acid molecules that can be controlled by irradiation with light and which we have developed in preliminary studies. This will now be taken significant steps forward by generating modular allosteric ribonucleic acid (RNA) assemblies that respond to light. These assemblies provide a generic solution and represent long-sought methods to complement the optogenetic toolbox. The aim of this project is the generation of allosteric molecules built from at least two RNA domains; one domain that binds to a soluble photoreceptor protein (PRP) in a light-dependent manner and a second RNA domain, whose protein inhibiting function in turn depends on the binding state of the PRP-recognizing part. We will construct light-responsive allosteric RNA assemblies that can be ubiquitously used in cells and in vivo, independent of specific model organisms, for optogenetic control and spatiotemporal analysis of endogenous, intracellular biomolecule function. The project is highly interdisciplinary and will open novel routes for biomolecule analysis in cells and in vivo. It has implications ranging from life sciences to optogenetics, and from combinatorial biochemistry to synthetic biology. As these new tools will be applicable by any scientist to analyse protein function at high spatiotemporal resolution, the project bears an enormous innovative potential.
Summary
Light-sensitive channel proteins have attracted much attention as functional molecules, as they possess unique conformations and functions depending on their respective irradiation states. Embedding these proteins in heterogeneous cellular frameworks enabled to gain light-control of cellular and in particular neuronal behaviour. However to date, optogenetic solutions that address endogenous, intracellular biomolecules in a universal fashion remain elusive. The project will apply design and selection strategies aiming at nucleic acid molecules that can be controlled by irradiation with light and which we have developed in preliminary studies. This will now be taken significant steps forward by generating modular allosteric ribonucleic acid (RNA) assemblies that respond to light. These assemblies provide a generic solution and represent long-sought methods to complement the optogenetic toolbox. The aim of this project is the generation of allosteric molecules built from at least two RNA domains; one domain that binds to a soluble photoreceptor protein (PRP) in a light-dependent manner and a second RNA domain, whose protein inhibiting function in turn depends on the binding state of the PRP-recognizing part. We will construct light-responsive allosteric RNA assemblies that can be ubiquitously used in cells and in vivo, independent of specific model organisms, for optogenetic control and spatiotemporal analysis of endogenous, intracellular biomolecule function. The project is highly interdisciplinary and will open novel routes for biomolecule analysis in cells and in vivo. It has implications ranging from life sciences to optogenetics, and from combinatorial biochemistry to synthetic biology. As these new tools will be applicable by any scientist to analyse protein function at high spatiotemporal resolution, the project bears an enormous innovative potential.
Max ERC Funding
1 992 438 €
Duration
Start date: 2014-05-01, End date: 2019-04-30
Project acronym POLLOX
Project ANAEROBIC POLLUTANT DEGRADATION WITH OXYGEN
Researcher (PI) Tillmann Joachim Lüders
Host Institution (HI) HELMHOLTZ ZENTRUM MUENCHEN DEUTSCHES FORSCHUNGSZENTRUM FUER GESUNDHEIT UND UMWELT GMBH
Call Details Consolidator Grant (CoG), LS9, ERC-2013-CoG
Summary Contamination with organic pollutants is widespread in nature and a notorious threat to our water resources. Two central paradigms are currently understood to control biodegradation in groundwater and sediments: (i) Redox gradients and interphases between compartments are hot-spots for contaminant breakdown, and (ii) biodegradation is primarily limited by local electron acceptor availability, in particular of oxygen. My group has published leading contributions to this understanding in recent years, especially in elucidating the ecology of anaerobic toluene degraders in aquifers.
This project now aims to question these established paradigms and to elaborate a ground-breaking new perspective of the role of molecular oxygen in pollutant degradation in anoxic compartments. The hypotheses at the heart of the project originate from a combination of own recent findings, partially inconsistent with the current understanding of process and degrader stratification at redox gradients. These are now interpreted in the light of exciting recent advances in the fields of electromicrobiology and oxygenic anaerobic respiration.
POLLOX postulates that oxygen-dependent degradation of pollutants in anaerobic compartments is possible by two unrecognised physiological adaptations of degraders. I want to verify the hypothesis that filamentous Desulfobulbaceae can anaerobically oxidise toluene via long-distance (1-2 cm) electron transfer to oxygen across redox gradients (aim 1). Furthermore, I postulate that monooxygenase-dependent toluene degraders, in absence of external oxygen, can be active via self-sustained production of oxygen by nitric oxide dismutation (aim 2). POLLOX proposes to perform targeted lab experiments and field surveys to verify both hypotheses and to elaborate the ecological niches in which respective degraders and processes are relevant in situ (aim 3). The generic mechanisms to be elaborated here have the potential to open new doors for bioremediation in the future.
Summary
Contamination with organic pollutants is widespread in nature and a notorious threat to our water resources. Two central paradigms are currently understood to control biodegradation in groundwater and sediments: (i) Redox gradients and interphases between compartments are hot-spots for contaminant breakdown, and (ii) biodegradation is primarily limited by local electron acceptor availability, in particular of oxygen. My group has published leading contributions to this understanding in recent years, especially in elucidating the ecology of anaerobic toluene degraders in aquifers.
This project now aims to question these established paradigms and to elaborate a ground-breaking new perspective of the role of molecular oxygen in pollutant degradation in anoxic compartments. The hypotheses at the heart of the project originate from a combination of own recent findings, partially inconsistent with the current understanding of process and degrader stratification at redox gradients. These are now interpreted in the light of exciting recent advances in the fields of electromicrobiology and oxygenic anaerobic respiration.
POLLOX postulates that oxygen-dependent degradation of pollutants in anaerobic compartments is possible by two unrecognised physiological adaptations of degraders. I want to verify the hypothesis that filamentous Desulfobulbaceae can anaerobically oxidise toluene via long-distance (1-2 cm) electron transfer to oxygen across redox gradients (aim 1). Furthermore, I postulate that monooxygenase-dependent toluene degraders, in absence of external oxygen, can be active via self-sustained production of oxygen by nitric oxide dismutation (aim 2). POLLOX proposes to perform targeted lab experiments and field surveys to verify both hypotheses and to elaborate the ecological niches in which respective degraders and processes are relevant in situ (aim 3). The generic mechanisms to be elaborated here have the potential to open new doors for bioremediation in the future.
Max ERC Funding
1 888 920 €
Duration
Start date: 2014-05-01, End date: 2019-04-30
Project acronym RNActivate
Project Optochemical control of cell fate by activation of mRNA translation
Researcher (PI) Andrea RENTMEISTER
Host Institution (HI) WESTFAELISCHE WILHELMS-UNIVERSITAET MUENSTER
Call Details Consolidator Grant (CoG), LS9, ERC-2017-COG
Summary Light is an excellent external regulatory element that can be applied to cells and organisms with high spatio-temporal precision and without interfering with cellular processes. Optochemical biology exploits small photo-responsive chemical groups to cage and activate or to switch biomolecular functions in response to light of a defined wavelength. Caged antisense agents have enabled down-regulation of gene expression with spatio-temporal control at the messenger-RNA (mRNA) level in vivo, however approaches for triggering translation of exogenous mRNA lack efficient turn-on effects. To explore the effects of conditional and transient ectopic gene expression in a developing organism it is vital to fully abrogate and restore translational efficiency.
The goal of this project is to bring eukaryotic mRNA under the control of light to trigger efficient ectopic translation with spatio-temporal resolution in cells and in vivo. To achieve this, eukaryotic mRNA will be photo-caged at its 5′ cap using a highly promiscuous methyltransferase capable of transferring very bulky moieties from synthetic analogs of the cosubstrate S-adenosylmethionine (AdoMet). A single 5′ cap modification will block translation of the respective mRNA. Its light-triggered removal will release unmodified capped RNA, which in cells will be efficiently remethylated to form the canonical 5′ cap resulting in uncompromised translation.
In addition to labeling and tracking subpopulations of cells, we will use our technology to control and to manipulate cell fate by locally producing proteins responsible for cell death, genome engineering, and cell migration. We will use cultured cells and one-cell stage zebrafish embryos that can be easily injected with mRNA to study the function of ectopic gene expression in early development. Our approach will overcome current limitations of photo-inducible mRNA translation and enable us to manipulate a developing organism at the molecular level.
Summary
Light is an excellent external regulatory element that can be applied to cells and organisms with high spatio-temporal precision and without interfering with cellular processes. Optochemical biology exploits small photo-responsive chemical groups to cage and activate or to switch biomolecular functions in response to light of a defined wavelength. Caged antisense agents have enabled down-regulation of gene expression with spatio-temporal control at the messenger-RNA (mRNA) level in vivo, however approaches for triggering translation of exogenous mRNA lack efficient turn-on effects. To explore the effects of conditional and transient ectopic gene expression in a developing organism it is vital to fully abrogate and restore translational efficiency.
The goal of this project is to bring eukaryotic mRNA under the control of light to trigger efficient ectopic translation with spatio-temporal resolution in cells and in vivo. To achieve this, eukaryotic mRNA will be photo-caged at its 5′ cap using a highly promiscuous methyltransferase capable of transferring very bulky moieties from synthetic analogs of the cosubstrate S-adenosylmethionine (AdoMet). A single 5′ cap modification will block translation of the respective mRNA. Its light-triggered removal will release unmodified capped RNA, which in cells will be efficiently remethylated to form the canonical 5′ cap resulting in uncompromised translation.
In addition to labeling and tracking subpopulations of cells, we will use our technology to control and to manipulate cell fate by locally producing proteins responsible for cell death, genome engineering, and cell migration. We will use cultured cells and one-cell stage zebrafish embryos that can be easily injected with mRNA to study the function of ectopic gene expression in early development. Our approach will overcome current limitations of photo-inducible mRNA translation and enable us to manipulate a developing organism at the molecular level.
Max ERC Funding
1 990 225 €
Duration
Start date: 2018-06-01, End date: 2023-05-31
Project acronym RNArepair
Project Site-directed RNA Editing to Manipulate RNA and Protein Function
Researcher (PI) Thorsten Stafforst
Host Institution (HI) EBERHARD KARLS UNIVERSITAET TUEBINGEN
Call Details Consolidator Grant (CoG), LS9, ERC-2014-CoG
Summary Enzymatically active RNA-guided proteins, like the RNA-induced silencing complex (RISC), are particularly versatile tools for the rationally programmed manipulation of genetic information. After successful re-addressing of various natural RNA-guided machineries it is now time to tackle the engineering of novel, user-defined tools. With this respect we have recently achieved the engineering of an RNA-guided adenosine-to-inosine RNA editing machinery. Since inosine is biochemically read as guanosine, A-to-I editing alters genomic information on the RNA-level and may potentially allow for the manipulation of RNA processing or protein function. We have already achieved to apply our RNA editing approach for the repair of several missense and nonsense point mutations on reporter and disease-related genes in vitro and demonstrated its applicability in mammalian cell culture.
Now, we want to push the method further towards application. To enable editing in oocytes, primary cells and neurons, we will establish to deliver the editing tool by lentiviral vectors and stabilized mRNAs. We further aim to create cell lines expressing the artificial editing machinery under conditional control. We will repair reporter genes in developing worm oocytes, and we want to reconstitute mutations that cause neuro-diseases. We also wish to establish new features including photocontrol and the application of editing to steer protein localization.
If successful, site-directed RNA editing will enable us to manipulate RNA and protein function in a yet unprecedented way. The ready introduction of point mutations into mRNAs without the need for genomic engineering may dramatically facilitate the study of protein function, disease mechanism and may even allow for the treatment of diseases based on personalized genetic information.
Summary
Enzymatically active RNA-guided proteins, like the RNA-induced silencing complex (RISC), are particularly versatile tools for the rationally programmed manipulation of genetic information. After successful re-addressing of various natural RNA-guided machineries it is now time to tackle the engineering of novel, user-defined tools. With this respect we have recently achieved the engineering of an RNA-guided adenosine-to-inosine RNA editing machinery. Since inosine is biochemically read as guanosine, A-to-I editing alters genomic information on the RNA-level and may potentially allow for the manipulation of RNA processing or protein function. We have already achieved to apply our RNA editing approach for the repair of several missense and nonsense point mutations on reporter and disease-related genes in vitro and demonstrated its applicability in mammalian cell culture.
Now, we want to push the method further towards application. To enable editing in oocytes, primary cells and neurons, we will establish to deliver the editing tool by lentiviral vectors and stabilized mRNAs. We further aim to create cell lines expressing the artificial editing machinery under conditional control. We will repair reporter genes in developing worm oocytes, and we want to reconstitute mutations that cause neuro-diseases. We also wish to establish new features including photocontrol and the application of editing to steer protein localization.
If successful, site-directed RNA editing will enable us to manipulate RNA and protein function in a yet unprecedented way. The ready introduction of point mutations into mRNAs without the need for genomic engineering may dramatically facilitate the study of protein function, disease mechanism and may even allow for the treatment of diseases based on personalized genetic information.
Max ERC Funding
1 808 200 €
Duration
Start date: 2015-08-01, End date: 2020-07-31
Project acronym STePLADDER
Project Solving The Pathway of LADDERane biosynthesis
Researcher (PI) Thomas Reinier Maxim BARENDS
Host Institution (HI) MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Call Details Consolidator Grant (CoG), LS9, ERC-2016-COG
Summary Ladderanes are highly unusual hydrocarbon moieties with linearly concatenated cyclobutane rings. They are found in the membranes of bacteria performing anaerobic ammonium oxidation (“anammox”). How these complicated molecules are produced is expected to involve both novel enzymes and specially evolved versions of known proteins, but the details remain enigmatic. How do these enzymes assemble such intricate carbon skeletons? How do they control the complex stereochemistry involved? How do they deal with the typical nonreactivity of hydrocarbons? And how do they overcome the ring strain inherent in these molecules? Answering these and other questions about the molecular mechanism of ladderane biosynthesis will open up new frontiers in enzymology.
I therefore propose to elucidate the pathway of ladderane biosynthesis.
I will use a synergistic approach, combining biochemistry, chemical biology and structural biology to arrive at a comprehensive view of ladderane biosynthesis in molecular detail. Heterologous expression of selected biosynthetic gene clusters will be used to study ladderane biosynthesis in vivo. Chemical biology will be used to load ladderanes and their intermediates onto carrier proteins to study their interactions with enzymes from the biosynthetic pathway in vitro. Protein crystallography will elucidate structures of enzymes and their complexes with ladderane-loaded carrier proteins. The knowledge accumulated will not only break new ground in enzymology, but also serve as a stepping stone for the use of these enzymes in bioinspired organic synthesis.
Summary
Ladderanes are highly unusual hydrocarbon moieties with linearly concatenated cyclobutane rings. They are found in the membranes of bacteria performing anaerobic ammonium oxidation (“anammox”). How these complicated molecules are produced is expected to involve both novel enzymes and specially evolved versions of known proteins, but the details remain enigmatic. How do these enzymes assemble such intricate carbon skeletons? How do they control the complex stereochemistry involved? How do they deal with the typical nonreactivity of hydrocarbons? And how do they overcome the ring strain inherent in these molecules? Answering these and other questions about the molecular mechanism of ladderane biosynthesis will open up new frontiers in enzymology.
I therefore propose to elucidate the pathway of ladderane biosynthesis.
I will use a synergistic approach, combining biochemistry, chemical biology and structural biology to arrive at a comprehensive view of ladderane biosynthesis in molecular detail. Heterologous expression of selected biosynthetic gene clusters will be used to study ladderane biosynthesis in vivo. Chemical biology will be used to load ladderanes and their intermediates onto carrier proteins to study their interactions with enzymes from the biosynthetic pathway in vitro. Protein crystallography will elucidate structures of enzymes and their complexes with ladderane-loaded carrier proteins. The knowledge accumulated will not only break new ground in enzymology, but also serve as a stepping stone for the use of these enzymes in bioinspired organic synthesis.
Max ERC Funding
1 753 125 €
Duration
Start date: 2017-06-01, End date: 2022-05-31
Project acronym StrainBooster
Project Enforced ATP wasting as a general design principle to rationally engineer microbial cell factories
Researcher (PI) Steffen Klamt
Host Institution (HI) MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Call Details Consolidator Grant (CoG), LS9, ERC-2016-COG
Summary One global challenge of humanity in the 21st century is the shift from a petrochemical to a bio-based production of chemicals and fuels. An enabling technology towards this goal is metabolic engineering which uses computational and experimental methods to construct microbial cell factories with desired properties. While it has been shown that genetically engineered microorganisms can, in principle, produce a broad range of chemicals, novel approaches to improve the performance of those strains are urgently needed to develop economically viable bioprocesses.
To this end, we propose a new metabolic design principle to rationally engineer cell factories with high performance. Supported by a recent pilot study, we postulate that suitable genetic interventions combined with mechanisms that burn (waste) an extra amount of ATP (e.g., by artificial futile cycles) will increase product yield and productivity of many microbial production strains. Key objectives of StrainBooster are therefore: (1) to use computational techniques and metabolic models to identify gene knockout strategies whose coupling with ATP wasting mechanisms can boost the performance of microbial strains and to prove in silico that those strategies exist for many combinations of substrates, products, and host organisms; (2) to develop genetic modules that can robustly increase ATP dissipation in the cell; and (3) to experimentally demonstrate the power of the proposed strategy for selected production processes with Escherichia coli. To reach these ambitious goals, an interdisciplinary approach will be pursued combining theoretical and experimental studies and making use of innovative methods from systems and synthetic biology.
If successful, StrainBooster will not only establish a new and ground-breaking strategy for metabolic engineering, it will also deliver novel computational tools and genetic parts facilitating direct application of the approach to design and optimize industrial fermentation processes.
Summary
One global challenge of humanity in the 21st century is the shift from a petrochemical to a bio-based production of chemicals and fuels. An enabling technology towards this goal is metabolic engineering which uses computational and experimental methods to construct microbial cell factories with desired properties. While it has been shown that genetically engineered microorganisms can, in principle, produce a broad range of chemicals, novel approaches to improve the performance of those strains are urgently needed to develop economically viable bioprocesses.
To this end, we propose a new metabolic design principle to rationally engineer cell factories with high performance. Supported by a recent pilot study, we postulate that suitable genetic interventions combined with mechanisms that burn (waste) an extra amount of ATP (e.g., by artificial futile cycles) will increase product yield and productivity of many microbial production strains. Key objectives of StrainBooster are therefore: (1) to use computational techniques and metabolic models to identify gene knockout strategies whose coupling with ATP wasting mechanisms can boost the performance of microbial strains and to prove in silico that those strategies exist for many combinations of substrates, products, and host organisms; (2) to develop genetic modules that can robustly increase ATP dissipation in the cell; and (3) to experimentally demonstrate the power of the proposed strategy for selected production processes with Escherichia coli. To reach these ambitious goals, an interdisciplinary approach will be pursued combining theoretical and experimental studies and making use of innovative methods from systems and synthetic biology.
If successful, StrainBooster will not only establish a new and ground-breaking strategy for metabolic engineering, it will also deliver novel computational tools and genetic parts facilitating direct application of the approach to design and optimize industrial fermentation processes.
Max ERC Funding
1 998 750 €
Duration
Start date: 2017-05-01, End date: 2022-04-30
