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Enforced ATP wasting as a general design principle to rationally engineer microbial cell factories

Periodic Reporting for period 2 - StrainBooster (Enforced ATP wasting as a general design principle to rationally engineer microbial cell factories)

Reporting period: 2018-11-01 to 2020-04-30

One global challenge of humanity in the 21st century is the shift from a petrochemical to a bio-based production of chemicals and fuels. An enabling technology towards this goal is metabolic engineering, which uses computational and experimental methods to construct microbial cell factories with desired properties. While it has been shown that genetically engineered microorganisms can, in principle, produce a broad range of chemicals, novel approaches to improve the performance of those strains are urgently needed to develop economically viable bioprocesses.
To this end, the goal of the StrainBooster project is to establish a new metabolic design principle to rationally engineer cell factories with superior performance. We postulate that suitable genetic interventions combined with mechanisms that burn (waste) an extra amount of ATP (e.g. by artificial futile cycles) will increase product yield and productivity of many microbial production strains. Key objectives of StrainBooster are therefore:
1. to use computational techniques and constraint-based metabolic models to identify gene knockout strategies whose coupling with ATP wasting mechanisms can boost the performance of microbial strains and to prove in silico that those strategies exist for many combinations of substrates, products, and host organisms;
2. to develop genetic modules that can robustly increase ATP dissipation in the cell;
3. to experimentally demonstrate the power of the proposed strategy for selected production processes with Escherichia coli.
To reach these ambitious goals, an interdisciplinary approach will be pursued, which combines theoretical and experimental studies and makes use of innovative methods from systems and synthetic biology. If successful, StrainBooster will not only establish a new and ground-breaking strategy for metabolic engineering, it will also deliver novel computational tools and genetic parts facilitating direct application of the approach to design and optimize industrial fermentation processes. The StrainBooster project consists of four (two theoretical and two experimental) interacting work packages reflecting its interdisciplinary nature (see figure).
We first developed several new computational methods and tools that have been / will be used for model-driven analysis and design of metabolic networks. Here, major achievements include new algorithmic approaches to compute metabolic engineering strategies [Venayak et al, 2018, Nature Comm] and to rank those computed strategies to facilitate the selection of most promising candidates for experimental implementation [Schneider and Klamt, 2019, Bioinformatics]. Those algorithmic developments have been integrated in our metabolic modelling software package CellNetAnalyzer, which is widely used in the community [von Kamp et al, 2017, Journal of Biotechnology].

We then used these and other metabolic modeling techniques to demonstrate the (theoretical) potential of StrainBooster’s metabolic design principle. As a key achievement, in a comprehensive computational study we could show that targeted interventions, that make production of the desired compound mandatory for cell growth and ATP synthesis, exist, in principle, for almost all metabolites producible in genome-scale metabolic models of five major production organisms [von Kamp and Klamt, 2017, Nature Communications]. These results are of fundamental importance for rational metabolic engineering in general as well as for the feasibility of the StrainBooster approach. Furthermore, we could demonstrate that ATP wasting mechanism as suggested by the StrainBooster approach can increase the productivity of two-stage processes, where growth and production are separated [Klamt et al., 2018, Biotechnology Journal].

The induction of artificial ATP wasting mechanisms in the cell is central for the metabolic design principle of StrainBooster. On the experimental side, we therefore developed a library of 11 genetic modules allowing static as well as dynamic activation of ATP wasting with different strengths. All modules rely on the expression of an ATP-hydrolyzing subunit of the ATPase as a very efficient and direct mechanism for uncoupled ATP hydrolysis and the functioning of these modules was confirmed in dedicated experiments [Boecker et al., 2019, Biotech J; Boecker et al., in preparation].

The ultimate goal of the whole project is to provide experimental showcases demonstrating the high potential of StrainBooster’s metabolic design strategy, i.e. that microbial cells with superior production performance can be constructed. In [Boecker et al., 2019, Biotech J] we presented a first such proof-of-principle. Taking the native fermentation products of E.coli as proxy for products of interest, we showed that induction of the ATPase (using the genetic modules mentioned above) leads to (i) higher metabolic activity and increased product formation even under anaerobic (growth-coupled) production conditions and (ii) that ATP wasting also significantly increases substrate uptake and productivity of growth-arrested cells which is vital for its use in two-stage processes. For example, the glucose uptake rate of 6.49 mmol/gCDW/h achieved with enforced ATP wasting is the highest value reported for non-growing E. coli cells. Hence, this study showed that enforced ATP wasting may improve yield and titer (in growth-coupled processes) as well as volumetric productivity (in two-stage processes). We are currently testing the effect of ATP wasting on the performance of dedicated production strains of E. coli (products: isobutanol, 2,3-butanediol) and in yeast (ethanol).
The StrainBooster approach for designing microbial cell factories (based on coupling ATP synthesis with product format in a first step followed by induction of a mechanism enforcing increased ATP dissipation) is new and unconventional. Indeed, feedback from colleagues we received at conferences etc. indicated that this approach appears non-intuitive to them and the promising results we obtained in our proof-of-principle study [Boecker et al., 2018, Biotechnology Journal]) were also unexpected to many peers. Moreover, several algorithmic techniques developed in WP2 and used in WP1 are new and allowed us, for example, to conduct the most comprehensive systematic computational screening of strain design strategies leading to results that are of fundamental importance for rational metabolic engineering [von Kamp and Klamt, 2017, Nature Communications; Venayak et al., 2018 Nature Communications]. We also believe that the tight connection of algorithmic and methodological developments and computational studies with experimental investigations (and verifications) is not standard in the field of metabolic engineering.

Having developed the required theoretical and experimental methods and provided a proof-of-principle study during the first half of StrainBooster, the second half of the project will now primarily focus on the development of showcases demonstrating the wide applicability of StrainBooster’s metabolic design approach to construct microbial cell factories (from different species) for the efficient production of different chemicals. We expect that we can deliver 4-5 such showcases at the end of the project.
Structure and work packages of the StrainBooster project.