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  • Periodic Reporting for period 1 - EmPowerPutida (Exploiting native endowments by re-factoring, re-programming and implementing novel control loops in Pseudomonas putida for bespoke biocatalysis)

EmPowerPutida Report Summary

Project ID: 635536
Funded under: H2020-EU.2.1.4.

Periodic Reporting for period 1 - EmPowerPutida (Exploiting native endowments by re-factoring, re-programming and implementing novel control loops in Pseudomonas putida for bespoke biocatalysis)

Reporting period: 2015-05-01 to 2016-10-31

Summary of the context and overall objectives of the project

Synthetic Biology (SynBio) is bound to be transformative in Industrial and Environmental Biotechnology. The powerful methodologies for high-throughput genome engineering, model-driven design and circuit construction, along with new concepts for bioprocess engineering enable the development of tailored biocatalys-based processes that would be otherwise impossible, unscalable or not economically feasible. SynBio has thus a great potential to foster the transition of a petrochemical to a bio-based economy. EmPowerPutida aims at developing a solid, versatile bacterial platform for whole-cell biocatalysis that is bound to take European chemical biotechnology into an unprecedented level of productivity and competitiveness. Accordingly, the project has the overarching goal of re-programming the lifestyle of Pseudomonas putida and designing a modular, streamlined bacterial platform for bespoke biocatalysis. This industrially driven project capitalizes on the outstanding metabolic endowment and stress tolerance capabilities of this versatile bacterium for the production of specialty and bulk chemicals.
Specifically, the project will build streamlined P. putida strains with improved ATP availability utilizing this power on demand, decoupled from growth. The well- characterized, streamlined and re-factored strain platform will offer easy-to-use plug-in opportunities for novel, DNA-encoded functions under the control of orthogonal regulatory systems. By drawing on a starkly improved, growth-uncoupled ATP-biosynthetic machinery, empowered P. putida strains will be able to produce industrial alcohols and their challenging gaseous derivatives using a novel, new-to-nature route starting from glucose, as well as b) new active ingredients for crop protection, such as high-value, ß-lactam-based secondary metabolites with a strong potential as a new herbicides.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project has progressed well and according to the plans over the first 18 months. A good part of the activities dealt the developing and implementations of various approaches for streamlining, re-factoring and re-engineering the P. putida chassis to render it more robust to toxic yet valuable products and amenable for process control, plugging in of designed circuits and subsequent bespoke biocatalysis. To this end, the genome of P. putida KT2440 was re-sequenced and fully re-annotated, yielded a number of new insights. A genome-scale metabolic model as a basis for model-driven design of the engineering activities was updated and reconciled with substantial physiological data. High-throughput recombineering tools, MetaBricks and a MAGE-like system were developed unto a large extent. A basis was set for optimization of traits industrially relevant traits such as yielding P. putida blind and deaf, making the strict-aerobe P. putida able to thrive under anoxic conditions, increase tolerance of P. putida to alcohols and implementing an efficient DNA-repair system.
Precise tools were developed to allow the conditional knock down of cellular functions with the aim in mind to disrupt cellular growth while maintaining a state of high metabolic activity, so that production pathways can continue to operate at high productivity. As this is expected to require the modulation of multiple functions, at least one of the methods has to be amenable to multiplexing and preferably suitable for employing it in a screen to identify such functions. The targeted tools include selective protein hydrolysis and repression of gene translation. In the first 18 months, we successfully developed an expression system with a switch-like characteristic to express highly toxic genes, which allows using selective proteases intracellularly without affecting cellular function prematurely. We also identified two small RNAs in P. putida which seem good candidates to be generalized into a sRNA knock down method for P. putida. Finally, we identified a variant of the AlkS regulatory protein that is sensitive to butanol, one of the target products of this project. Therefore, all activities towards a functionally flexible and multi-plexable conditional knockdown platform in P. putida can record major progress.
In addition to the groundwork related to optimization and modularization of the P. putida chassis, we carried out activities laying the basis to enable accessing the production of industrial alcohols and derivatives, and high-value secondary metabolites. Importantly, progress was made regarding the ATP-dependent biosynthetic engineering and implementation of non-natural pathways.
We have worked on developing and implementing novel engineering circuits in P. putida, to enable novel control possibilities, including the build-up of an ATP sensor to sense changing supply of ATP on demand and sRNA-based regulation. In parallel, a detailed dynamic model has been formulated to describe controlled loops of energy supply. The model is to be used for analysis and design of strain construction and, in conjunction with the genome-scale metabolic model, to assess the interplay between the circuits developed and the chassis.
First reference fermentations were made to obtain performance data of Pseudomonas putida in batch and continuous cultivations as well as under defined stress conditions. This will set the basis for subsequent validation and scaling-up of the processes herein developed.
A data management strategy was developed and implemented in the project to ensure findability, accessibility, interoperabiity and reproducibility (FAIR) of Data, Operation procedure and models (DOM). Furthermore, a series of activities geared at increasing awareness and dealing with biosafety were implemented. Through dissemination and training, EmPowerPutida has been engaging with society at large and including all its activities in a wider setting than that of industrial biotechnolog

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The game-changing innovations brought in – in particular the uncoupling of ATP-synthesis and production from growth - will provide strong versatility, enhanced efficiency and efficacy to the production processes, thereby overcoming current bottlenecks, matching market needs and fostering high-level research growth and development. The technological and market potential of EmPowerPutida concept is very substantial. The achievement of objectives and exploitation of technologies will not only provide a high impact in the partners of the consortium but also, and more drastically, contribute to boost the global competitiveness of the European industries (reduction of the cost of chemicals and environmental benefits) and to create new economic opportunities. More specifically, the impact on the EU industrial competitiveness can be derived from the added value of the EmPowerPutida to the chemical industry. In the short term period after project completion, EU industry will beneficiate from the bio-production capacity of the targeted chemicals using novel new-to-nature route starting from renewable glucose sources –overcoming price fluctuation caused by temporal shortages of the established cracker products based on crude oil.

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