Synthetic bacterial lipid rafts to optimize industrial bioprocesses

Periodic Reporting for period 3 - Rafts4Biotech (Synthetic bacterial lipid rafts to optimize industrial bioprocesses)

Période du rapport: 2020-01-01 au 2021-06-30

Genetic tractability of bacterial cells allows generating synthetic microbial chassis platforms (SMCPs) with remarkable biotechnological applications but their functionality currently faces important off-genome limitations
due to deficient protein-protein interactions, unfavorable protein stoichiometry or generation of toxic intermediates that ultimately compromise the industrial production processes. To solve this problem, Rafts4Biotech project will take advantage of our recent discovery, that bacteria are able to organize subcellular membrane compartments similar to the so-called lipid rafts of eukaryotic cells, to improve/protect specific cellular processes. Rafts4Biotech project will engineer bacterial cells to confine biotechnologically relevant reactions into bacterial lipid rafts to optimize their stoichiometry and protect cells from undesirable metabolic interferences. Hence, the Rafts4Biotech project will produce new generation reliable and robust SMCPs in which industrial production processes are confined in bacterial lipid rafts, released from their classical off-genome limitations and optimized for industrial production. Moreover, this concept can be applied to many prokaryotes, since lipid rafts happens to occur in many bacterial species. Based on this versatility, Rafts4Biotech project will use two biotechnologically relevant biosystems, Bacillus subtilis and Escherichia coli, to engineer synthetic bacterial lipid rafts to optimize the performance of three challenging biochemical processes in the fields of pharmaceutical, cosmetics and feed industrial sectors. To achieve this, Rafts4Biotech consortium combines different expertise in synthetic biology, systems biology and mathematical modeling and it includes a number of SMEs that will actively work in this project and will translate this technology into market application. The technology developed by Rafts4Biotec will optimize multistep industrial processes and invigorate European research.

Societal impact: R4B project will develop high value-added products that will contribute to create a positive opinion in the society about synthetic and systems biology. Furthermore, Rafts4Biotech does not plan to release any construct to the open environment and therefore, we anticipate good reception of this project in all stakeholders and societal players of industrial biotechnology and will contribute to generate a better public opinion on biotechnology and synthetic biology.

Overall Objective: R4B will develop a new, versatile generation of microbial chassis to confine industrially relevant reactions in subcellular membrane compartments recently discovered and named bacterial lipid rafts, which will allow the fine regulation of the industrial processes, their optimal performance and their isolation from cellular metabolism to prevent toxic reactivity.

Rafts4Biotech is a 48-months duration research project that will be developed by a well-balanced consortium constituted by 3 RTOs, 4 Universities and 5 SMEs (See page 1). The methodology required to develop Rafts4Biotech proposal is based on an integrated systems biology approach combined with a number of distinct disciplines, such as mathematical modeling, enzymology and synthetic biology approaches. This multifaceted disciplinary aspect of Rafts4Biotech consortium is consequence of our conviction to develop bacterial synthetic lipid rafts as an innovative technology with broad industrial applications. The below described work packages (WPs) of this proposal aim to demonstrate the versatility of our technology by addressing three important biotechnological conundrums that are caused by the current metabolic limitations that suffer conventional microbial chassis: 1) Engineering cell factories to produce novel lipophilic antibiotics difficult to produce insofar in laboratory conditions (Pharma-SC). 2) Implementing existing cell factories to produce cosmetic additives that currently show low production yields and high production costs (Cosmetics-SC) and 3) Developing more robust and resistant cell factories to eliminate industrial pollutants from food and drinking sources (Feed-SC).

The genetic tractability of bacterial cells allows generating synthetic microbial chassis platforms (SMCPs) expressing intricate genetic circuits with remarkable biotechnological applications. Despite their genetic versatility, the correct functionality of SMCPs currently faces important off-genome limitations due to deficient protein-protein interactions, unfavorable protein stoichiometry and generation of toxic intermediates that interfere with the metabolism and ultimately compromise the industrial production processes. Overcoming these constrains is one of the most important challenges of synthetic biology for the implementation of artificial biosystems. Rafts4Biotech project aims to solve this hurdle. The component of innovation of Rafts4Biotech project is based on our recent discovery, that bacteria are able to organize specific subcellular membrane compartments, similar to the so-called lipid rafts of eukaryotic cells, to improve the efficiency of specific cellular processes and protect them from cross-reactions outside bacterial lipid rafts. Based on this discovery, it is possible to engineer bacterial cells to confine biotechnologically relevant reactions into lipid rafts to optimize their stoichiometry and protect cells from undesirable metabolic interferences. The Rafts4Biotech project will implement this novel technology and will produce a new generation of reliable and robust SMCPs in which industrial production processes are confined in bacterial lipid rafts. Using this new technology, SMCPs will be released from their classical off- genome limitations and will be optimized for industrial production. Moreover, these new artificial biosystems can select for and against a plethora of biological interactions and produce extremely tunable microbial chassis for the production of a large and diverse group of value-added products. Furthermore, limiting the interactions with cellular metabolism will increase robustness of the cell factories because it will protect cells from undesirable reactions and thus the accumulation of toxic intermediates.

Importantly, the scientific concept that we will develop in Rafts4Biotech proposal can be applied to an enormous diversity of prokaryotes that are conventionally or non-conventionally used as microbial chassis in industrial settings. This is because the existence of bacterial lipid rafts has been detected in almost all bacterial species. Based on the versatility of this new technology, the Raft4Biotech project will use two biotechnologically relevant microbial systems, the Gram-positive bacterium Bacillus subtilis and the Gram- negative bacterium Escherichia coli, to engineer synthetic bacterial lipid rafts to optimize the performance of three challenging biochemical processes of growing biotechnological interest, which will be used as study cases for the completion of this proposal and are detailed below:

1) Producing novel antibiotics to fight resistant infections (Pharma Study Case or Pharma-SC)
2) Implementing biochemical pathways of commercial value (Cosmetics Study Case or Cosmetics-SC)
3) Improving microbial chassis to resist harsh industrial processes (Food Study Case or Feed-SC)

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Periodic Reporting for period 3 - Rafts4Biotech (Synthetic bacterial lipid rafts to optimize industrial bioprocesses)

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