We successfully developed and implemented 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, CRISPRi and a MAGE-like system were developed. A basis was set for optimization industrially relevant traits such as rendering 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 of disrupting cellular growth while maintaining a state of high metabolic activity, so that production pathways can continue to operate at high productivity. 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 small RNAs in P. putida that underpin a sRNA knock down method for P. putida.
We developed and implemented 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 was 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.
New synthetic routes to industrially interesting unsaturated products isobutene and butadiene have been established based on renewable resources. A route to crotyl alcohol, a key intermediate, was cloned for use in P. putida. By the implementation of the cascade into P. putida, enzymes degrading crotyl alcohol were identified as targets for potential knock-outs, further widening the application spectrum of the bacterium in future industrial processes. The production of isobutene and butadiene is to date mainly achieved by steam cracking. Our newly developed enzymatic routes play an important role in uncoupling production of these widely used compounds from fossil resources.
Applying a dedicated reactor setup, we found that P. putida KT2440 minimizes the large-scale induced stress stimuli to a minimum of transcriptional and metabolic burden on the cell. It does so by rapidly degrading large PHA pools under sudden glucose starvation such that intracellular ATP pools can completely recover. Furthermore, process development for isobutanol production continued identifying intracellular NADPH shortage as a key limiting step hampering further performance improvements.
Despite successful enzyme engineering of LinD (linalol dehydratase-isomerase) catalyzing the reaction of crotyl alcohol to butadiene, producing butadiene from glucose in P. putida KT2440 was not successful yet which prevented any bioprocess development. Also, for isobutene no production strain could be established. For tabtoxin, although its heterologous production could be successfully shown in P. putida, titers were below the threshold that would justify further bioprocess development. The biosynthetic route to methacrylates was established in P. putida KT2440., with the resulting strain being able to produce methacrylates from glucose.
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 engaged with society at large and including all its activities in a wider setting than that of industrial biotechnology only. Indeed, we are highly conscious that the rise of synthetic biology has raised great concern, and for this reason we are undertaking different actions to publicize what SynBio really is and what contributions can make to society.
