Periodic Reporting for period 4 - StrainBooster (Enforced ATP wasting as a general design principle to rationally engineer microbial cell factories)
Berichtszeitraum: 2021-11-01 bis 2022-12-31
One global challenge of humanity in the 21st century is the shift from a petrochemical to a sustainable production of chemicals and fuels. Bio-based production processes are one major building block for this transition. While it has been shown that genetically engineered microorganisms can, in principle, produce a broad range of chemicals, novel approaches to improve their performance are urgently needed to develop economically viable bioprocesses.
To this end, the StrainBooster project aimed to establish a novel powerful strategy for the rational engineering of cell factories with superior performance. We postulated that suitable genetic interventions combined with mechanisms that burn (waste) an extra amount of ATP (enforced ATP wasting = EAW) will increase product yield and productivity of many microbial production strains. Key objectives of the project were
1. to develop and use computational techniques to prove in silico that EAW can boost the performance of bioprocesses for many combinations of substrates, products, and host organisms;
2. to develop genetic modules for a flexible induction of ATP wasting in the cell;
3. to experimentally demonstrate the power of the proposed strategy for selected microbial production processes.
The project followed a an interdisciplinary approach combining theoretical and experimental studies and making use of innovative methods from systems and synthetic biology.
After the end of the project, we can conclude that StrainBooster was very successful and reached its central goals. First of all, basic theoretical and experimental investigations laid the foundations to implement EAW as a novel metabolic engineering strategy. In several application examples we could then validate StrainBooster’s central hypothesis and demonstrate the high potential of EAW to enhance the production performance of microbial strains. For example, synthesis rates of the platform chemcial 2,3-butanediol by E. coli could be increased by up to 500% and the product yield by 45%. The project also resulted in the development of novel computational tools to design and optimize industrial fermentation processes.
To this end, the StrainBooster project aimed to establish a novel powerful strategy for the rational engineering of cell factories with superior performance. We postulated that suitable genetic interventions combined with mechanisms that burn (waste) an extra amount of ATP (enforced ATP wasting = EAW) will increase product yield and productivity of many microbial production strains. Key objectives of the project were
1. to develop and use computational techniques to prove in silico that EAW can boost the performance of bioprocesses for many combinations of substrates, products, and host organisms;
2. to develop genetic modules for a flexible induction of ATP wasting in the cell;
3. to experimentally demonstrate the power of the proposed strategy for selected microbial production processes.
The project followed a an interdisciplinary approach combining theoretical and experimental studies and making use of innovative methods from systems and synthetic biology.
After the end of the project, we can conclude that StrainBooster was very successful and reached its central goals. First of all, basic theoretical and experimental investigations laid the foundations to implement EAW as a novel metabolic engineering strategy. In several application examples we could then validate StrainBooster’s central hypothesis and demonstrate the high potential of EAW to enhance the production performance of microbial strains. For example, synthesis rates of the platform chemcial 2,3-butanediol by E. coli could be increased by up to 500% and the product yield by 45%. The project also resulted in the development of novel computational tools to design and optimize industrial fermentation processes.
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.
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.
The StrainBooster project enabled us to establish EAW as a new metabolic engineering strategy to enhance the metabolic capabilities of microbial species in bioproduction processes. According to our findings, , if product synthesis in the cellular metabolism is coupled with net ATP formation, EAW is especially useful to increase the product yield in growth-coupled production processes or to boost volumetric productivity in the pro¬duc¬tion phase of two-stage processes. Feedback from colleagues indicated that the strategy of enforced ATP wasting appears rather non-intuitive and the convincing results we obtained in our proof-of-principle studies were unexpected to many peers. Our approach demonstrates the potential of synthetic biology to boost bio-based manufacturing with many possible applications in the biotech industry. Moreover, several algorithmic techniques developed and used in the project 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 in general, and for our ATP wasting approach in particular.