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This project, which translates “an idea to application”, builds upon a series of national and international activities.

We aim to engineer the lifestyle of Pseudomonas putida to generate a tailored, re-factored chassis with highly attractive new-to-nature properties, thereby opening the door to the production of thus far non-accessible compounds.

The guiding idea is to embed optimised biosynthetic pathways into an optimised chassis offering new functionalities.

By using a suite of advanced genetic tools, the properties of Pseudomonas putida – in particular redox balance and ATP production - will be enhanced while deleterious or useless qualities for the process will be removed.

The well-characterised, streamlined and re-factored strain platform will thus offer easy-to-use plug-in opportunities for novel, DNA-encoded functions under the control of orthogonal regulatory systems. The applicability of this microbial platform will be demonstrated by two case studies, namely on specialty chemicals for crop protection and biofuel molecules.

Our project is structured along 4 blocks and based on 8 work packages (WPs) as follows:

 

A - Lifestyle Engineering

De-bugging and enhancing the functional frame (WP1)

We focus on various approaches for streamlining, re-factoring and re-engineering the Pseudomonas putida chassis to render it more robust to products and amenable for process control, plugging in designed circuits and subsequent bespoke biocatalysis.

Lead: Agencia Estatal Consejo Superior de Investigaciones Científicas, Prof. Víctor de Lorenzo

Partners: Wageningen University, Abengoa Research SL

Harnessing the benefits of Lifestyle Engineering (WP2)

We aim to identify a set of conditional molecular interventions that lead, in response to an external signal, to the decoupling of cellular growth and product formation.

For this, we will develop and implement a multiplex-capable cellular intervention strategy for Pseudomonas putida based on:

  1. conditional expression of specific proteases for targeting cognate enzymes of interest
  2. synthetic DNA-oligonucleotides (conditional on the success of WP1), and
  3. sRNA based post-transcriptional control of gene expression

Lead: Eidgenoessische Technische Hochschule Zuerich, Prof. Sven Panke

Partners: Wageningen University, Agencia Estatal Consejo Superior de Investigaciones Científicas, Universidade Nova de Lisboa

B - Harnessing the Lifestyle Benefits

Engineering biocatalytic activities (WP3)

WP 3 opens the door to the targeted compounds by the optimization and modularization of the metabolism in the Pseudomonas putida chassis as well as the ATP-dependent biosynthetic engineering and implementation of non-natural pathways. Consequently, native pathways to these molecules will be de-bottlenecked rigorously and new routes successfully engrafted. Metabolic engineering and synthetic biology tools will be used.

Lead: Universitaet Stutttgart, Prof. Dr. Bernhard Hauer

Partners: Ingenza Ltd., BASF SE

Engineering circuits (WP4)

This WP focuses on the creation and implementation of novel engineering circuits in Pseudomanas putida thereby engrafting novel control possibilities, among them the supply of ATP. We will:

  1. develop novel regulatory sRNA
  2. reengineer ATP availability
  3. quantify regulatory circuits in vivo and embed related models in genome-scale modeling approaches

Lead: Universitaet Stuttgart, Prof. Dr. Ralf Takors

Partners: Wageningen University, Universidade Nova de Lisboa

C - From the Lab to Process Development

Validating and scaling-up the process (WP5)

WP5 focuses on the lab-to-production bioprocess development of the fermentation processes for the targeted compounds strictly considering industrial constraints for scale-up and production. Labscale experiments will be performed providing the data for realistic upscaling scenarios. The later will be performed as soon as economic and safety threshold will be passed successfully.

Lead: BASF SE, Dr. Andrea Herold

Partners: Universitaet Stuttgart, Abengoa Research SL

Exploitation and market-oriented dissemination (WP6)

The task in WP6 is to ensure that all project results are formulated and compiled into a protectable form. Therefore a market-oriented dissemination strategy will be developed to enable widespread publication of the project results among industry stakeholders.

The Consortium’s ability to exploit the project's results through knowledge sharing, documentation and training will be ensured.

The generated knowledge and technology will be used to influence decision-making institutions, raising end-user investments and to create awareness around the product development leading to a high socio-economic impact.

Lead: University of Wageningen, Prof. Vítor Martins dos Santos

Partners: Universitaet Stuttgart, Eidgenoessische Technische Hochschule Zuerich, Ingenza Ltd., LifeGlimmer GmbH, Abengoa Research SL, BASF SE

D - Coordination, Communication and ELSI

Ethical, Legal & Social Implications (ELSI) – Biosafety (WP7)

The tasks related to WP7 are:

  1. to implement overall awareness as well as assessment for ELSI aspects
  2. to contribute to the ethics debate in the synthetic biology community and in the public
  3. to implement and assess biosafety and biosecurity aspects in the project to maximise the benefits of scientific research while minimising the potential for societal harm

Lead: LifeGlimmer GmbH, Dr. Babette Regierer

Partners: Wageningen University, Agencia Estatal Consejo Superior de Investigaciones Científicas, Universidade Nova de Lisboa, Ingenza Ltd., Abengoa Research SL

Coordination, Management and Communication (WP8)

To ensure that all project objectives are met, WP8 will:

  1. establish and provide appropriate overall administrative and financial management
  2. monitor and report about project progress and achievements of the project objectives, evaluation and risk management
  3. establish and maintain communication within the consortium, with the EC bodies and relevant external bodies
  4. establish decision-making processes, undertake conflict management and organise consortium meetings
  5. establish and maintain a common exploitation and dissemination strategy
  6. advise and guide the participants to comply with the EU regulations and with their contractual and legal requirements

Lead: Wageningen University, Prof. Vitor Martins dos Santos

Partner: LifeGlimmer GmbH

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This project has received funding from the European Union’s Horizon 2020

research and innovation programme under grant agreement No 635536.

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