The StrainBooster project consisted of four (two theoretical and two experimental) interacting work packages, which reflected its interdisciplinary nature.
We first developed several new computational methods that can be used for model-driven analysis and design of metabolic networks. These include methods to compute metabolic engineering strategies based on our previously developed minimal cut set framework. The new algorithms have been integrated in our metabolic modeling software packages CellNetAnalyzer, which is widely used in the community, and in its new Python variant CNApy. We then used these and other metabolic modeling techniques to demonstrate the (theoretical) potential of EAW as metabolic design principle. As a key achievement in this direction, in a comprehensive computational study we could show that targeted interventions, that couple growth with product synthesis, exist for almost all metabolites producible by several major production organisms. Importantly, this growth coupling can be achieved by coupling ATP formation with product synthesis meaning that EAW may indeed increase productivity of these strains designs. These theoretical results are of fundamental importance for rational metabolic engineering in general as well as for the feasibility of the StrainBooster approach in particular. In another theoretical study we could demonstrate that the enforced ATP wasting advocated by the StrainBooster approach can increase the productivity of two-stage processes, where growth and production are separated.
The induction of ATP wasting mechanisms at different levels in the cell is essential for the metabolic design principle proposed by StrainBooster. We therefore developed a library of plasmids expressing the genes of the cytosolic F1-portion of the ATPase (which catalyzes uncoupled ATP hydrolysis). The ATPase genes are under control of a promoter inducible either with m-toluate or with IPTG. We also characterized oxygen-dependent promotors for dynamic activation of the ATPase genes when switching from aerobic to anaerobic conditions in two-stage processes. Hence, a library of vectors is now available, which allow static as well as dynamic activation of ATP wasting with different strengths. Several of these modules have been used in the application examples described below.
The ultimate goal of the StrainBooster project was to provide experimental showcases demonstrating the high potential of EAW to enhance the performance of microbial cell factories in bio-based production processes. In first studies with E. coli we could show that EAW increases the specific for¬mation rates of fermentation products during anaerobic growth by 20-30 % and the product yield by 7-10 %. Even more pronounced was the result under growth arrest (relevant for two-stage processes): the measured glucose uptake rate of 10.46 mmol/gCDW/h achieved with EAW is 450% higher than in the wild-type strain and the highest ever reported for growth-arrested E. coli cells. Specific and volumetric synthesis rates of fermentation products were similarly increased by EAW. In a next step, we used EAW to enhance the conversion of glucose to the platform chemical 2,3-butanediol (2,3-BDO) by an engineered E. coli strain. Under all cultivation conditions tested, we found that EAW increases the specific glucose uptake and 2,3-BDO synthesis rates drastically (sixfold and tenfold, respectively). Likewise, the 2,3-BDO yield was increased by up to 45 %. We also tested the effect of EAW in yeast (S. cerevisiae) on ethanol synthesis and found that, under anaerobic conditions, the ethanol yield is significantly improved (from 79 % to 87 % of the maximum yield). Furthermore, under growth-decoupled operation, EAW increased the specific and volumetric productivity of ethanol by over 100 % highlighting again the potential of EAW for enhancing two-stage processes.
Overall, StrainBooster’s central goals could be reached and our strain design principle is now ready for applications in industrial bioprocesses. Testing this strategy with biotech companies is subject of a submitted PoC ERC grant.
