Periodic Reporting for period 1 - PROHITS (Prokaryote proteomics at high temperature for single cells)
Periodo di rendicontazione: 2024-01-01 al 2025-12-31
A sustainable climate and an ecosystem-friendly economy demands novel "cell factories", in which organisms convert renewable biomass into various biomaterials that would otherwise be derived from fossil fuels or (harsh) chemistry. Especially promising is the use of thermophilic bacteria and archaea that operate at high temperatures, as this can enable more efficient production processes. However,we currently know very little about the cellular protein machinery (or proteome) of thermophiles, which limits our ability to efficiently use them in cell factories. The PROHITS project therefore creates a network for ten doctoral candidates to develop mass spectrometry (MS) driven proteomics approaches to study the in vivo proteome of thermophiles, and to apply the obtained insights to cell factory development.
Goal 1: Perform proteomics on single prokaryotic cells using advanced microfluidics, MS instrumentation and data analysis, to observe how proteomes vary in individual cells.
Goal 2: Enable thermal proteome profiling approaches for thermophiles, to observe how their proteomes respond to temperature changes.
Goal 3: Apply the proteomics insights for predicting how in vivo proteins respond to high temperatures, and create more detailed genome-scale models of thermophiles.
This information is used to improve an existing cell factory that produces lipids for medicine encapsulation, and to expand knowledge of thermophiles that can be used as cell factories. To achieve this, PROHITS creates an integrated pipeline of experimental and computational approaches, built within a collaborative network of academics and industry.
The doctoral candidates will obtain comprehensive and highly complementary skills, ranging from cutting-edge proteomics analysis, over data analysis and prediction, to systems biology approaches to optimise cell factories, which will enable them as future leaders of a thriving and forward-looking European bioeconomy.
Goal 1: Perform proteomics on single prokaryotic cells using advanced microfluidics, MS instrumentation and data analysis, to observe how proteomes vary in individual cells.
Goal 2: Enable thermal proteome profiling approaches for thermophiles, to observe how their proteomes respond to temperature changes.
Goal 3: Apply the proteomics insights for predicting how in vivo proteins respond to high temperatures, and create more detailed genome-scale models of thermophiles.
This information is used to improve an existing cell factory that produces lipids for medicine encapsulation, and to expand knowledge of thermophiles that can be used as cell factories. To achieve this, PROHITS creates an integrated pipeline of experimental and computational approaches, built within a collaborative network of academics and industry.
The doctoral candidates will obtain comprehensive and highly complementary skills, ranging from cutting-edge proteomics analysis, over data analysis and prediction, to systems biology approaches to optimise cell factories, which will enable them as future leaders of a thriving and forward-looking European bioeconomy.
Below describes the status of PROHITS at the point where the doctoral candidates have been working for 1 year and ~4 months.
Goal 1: The separation of single prokaryotes via microfluidics has been achieved. Concurrently, both experimental and computational analysis mass spectrometry (MS) methodology is being developed to obtain sufficient proteomics signal for these single cells, which contain very little protein. To ensure sufficient protein for mass spectrometry analysis whilst the MS methodology is being developed, alternative strategies are being explored such as mini-bulk, where hundreds of cells are being collected.
Goal 2: Standard cultivation and MS sample preparation protocols are available for six thermophilic species. MS Thermal Protein Profiling (TPP) experiments are being performed on these species to collect temperature-dependent data for their proteomes. In parallel, a full TPP data analysis pipeline has been developed that enables users without computational knowledge to analyse TPP data. The experiments have already started to further reveal the role of the thermosome, an archaeal chaperonin protein complex, in stabilising proteins at higher temperatures.
Goal 3: A framework for the prediction of protein melting temperatures is being set up that will be able to incorporate the full data from TPP experiments for training. A genome-scale metabolic model (GEM) was developed and experimentally validated for the S. acidocaldarius species. Finally, novel archaeal hosts were investigated for their potential for tetraether lipid production.
In the next stage of the project, these efforts will be further incorporated and developed; the single cell proteomics will enable observing variation in proteins that is relevant for development of GEMs and understanding how proteins are stabilised in the cell, as well as serving as validation for developed cell factories. The TPP data will be used for developing and fine-tuning a prediction framework for the thermal stability of single proteins in their in vivo context, as well as contributing to our understanding of the thermal response of proteomes, including the role of chaperonins. The collected proteome data on different species will further contribute to improving the GEMs developed within PROHITS.
Goal 1: The separation of single prokaryotes via microfluidics has been achieved. Concurrently, both experimental and computational analysis mass spectrometry (MS) methodology is being developed to obtain sufficient proteomics signal for these single cells, which contain very little protein. To ensure sufficient protein for mass spectrometry analysis whilst the MS methodology is being developed, alternative strategies are being explored such as mini-bulk, where hundreds of cells are being collected.
Goal 2: Standard cultivation and MS sample preparation protocols are available for six thermophilic species. MS Thermal Protein Profiling (TPP) experiments are being performed on these species to collect temperature-dependent data for their proteomes. In parallel, a full TPP data analysis pipeline has been developed that enables users without computational knowledge to analyse TPP data. The experiments have already started to further reveal the role of the thermosome, an archaeal chaperonin protein complex, in stabilising proteins at higher temperatures.
Goal 3: A framework for the prediction of protein melting temperatures is being set up that will be able to incorporate the full data from TPP experiments for training. A genome-scale metabolic model (GEM) was developed and experimentally validated for the S. acidocaldarius species. Finally, novel archaeal hosts were investigated for their potential for tetraether lipid production.
In the next stage of the project, these efforts will be further incorporated and developed; the single cell proteomics will enable observing variation in proteins that is relevant for development of GEMs and understanding how proteins are stabilised in the cell, as well as serving as validation for developed cell factories. The TPP data will be used for developing and fine-tuning a prediction framework for the thermal stability of single proteins in their in vivo context, as well as contributing to our understanding of the thermal response of proteomes, including the role of chaperonins. The collected proteome data on different species will further contribute to improving the GEMs developed within PROHITS.
At this stage of the project, there are already several results beyond previous state of the art:
- Reliable single cell prokaryote separation by microfluidics
- MS analysis of very low protein concentrations
- User-friendly computational framework for TPP data analysis
- A well-developed GEM for S. acidocaldarius
Their further development will be discussed during the next stage of the project.
- Reliable single cell prokaryote separation by microfluidics
- MS analysis of very low protein concentrations
- User-friendly computational framework for TPP data analysis
- A well-developed GEM for S. acidocaldarius
Their further development will be discussed during the next stage of the project.