In silico assessment of cell-free systems.

Cell-free extract (CFX)-derived biocatalytic systems are usually embedded in a complex metabolic network, which makes chemical insulation of the production system necessary by removing enzymatic connections. While insulation can be performed by different methods, the identification of potentially disturbing reactions can become a rather lengthy undertaking requiring extensive experimental analysis and literature review. Therefore, a tool for network topology analysis in cell-free systems was developed based on genome scale metabolic models. Genome scale metabolic models define a potential network topology for living cells, and can be adapted to the characteristics of cell-free systems by: (i) removal of compartmentalization, (ii) application of different objective functions, (iii) enabling the accumulation of all metabolites, (iv) applying different constraints for substrate supply, and (v) constraining the reaction space through cofactor availability, microarray data, feasible reaction rates, and thermodynamics. The resulting computational tool successfully predicted for Escherichia coli-derived CFXs a previously identified undesired pathway for dihydroxyacetone phosphate (DHAP) production from adenosine phosphates. The tool was then applied to the identification of potentially interfering pathways to further insulate a DHAP-producing multi-enzyme system based on CFX.

Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis.

Recruiting complex metabolic reaction networks for chemical synthesis has attracted considerable attention but frequently requires optimization of network composition and dynamics to reach sufficient productivity. As a design framework to predict optimal levels for all enzymes in the network is currently not available, state-of-the-art pathway optimization relies on high-throughput phenotype screening. We present here the development and application of a new in vitro real-time analysis method for the comprehensive investigation and rational programming of enzyme networks for synthetic tasks. We used this first to rationally and rapidly derive an optimal blueprint for the production of the fine chemical building block dihydroxyacetone phosphate (DHAP) via Escherichia coli's highly evolved glycolysis. Second, the method guided the three-step genetic implementation of the blueprint, yielding a synthetic operon with the predicted 2.5-fold-increased glycolytic flux toward DHAP. The new analytical setup drastically accelerates rational optimization of synthetic multienzyme networks.

Engineering in complex systems.

The implementation of the engineering design cycle of measure, model, manipulate would drastically enhance the success rate of biotechnological designs. Recent progress for the three elements suggests that the scope of the traditional engineering paradigm in biotechnology is expanding. Substantial advances were made in dynamic in vivo analysis of metabolism, which is essential for the accurate prediction of metabolic pathway behavior. Novel methods that require variable degrees of system knowledge facilitate metabolic system manipulation. The combinatorial testing of pre-characterized parts is particularly promising, because it can profit from automation and limits the search space. Finally, conceptual advances in orthogonalizing cells should enhance the reliability of engineering designs in the future. Coupled to improved in silico models of metabolism, these advances should allow a more rational design of metabolic systems.

Exploiting cell-free systems: Implementation and debugging of a system of biotransformations.

The orchestration of a multitude of enzyme catalysts allows cells to carry out complex and thermodynamically unfavorable chemical conversions. In an effort to recruit these advantages for in vitro biotransformations, we have assembled a 10-step catalytic system-a system of biotransformations (SBT)-for the synthesis of unnatural monosaccharides based on the versatile building block dihydroxyacetone phosphate (DHAP). To facilitate the assembly of such a network, we have insulated a production pathway from Escherichia coli's central carbon metabolism. This pathway consists of the endogenous glycolysis without triose-phosphate isomerase to enable accumulation of DHAP and was completed with lactate dehydrogenase to regenerate NAD(+). It could be readily extended for the synthesis of unnatural sugar molecules, such as the unnatural monosaccharide phosphate 5,6,7-trideoxy-D-threo-heptulose-1-phosphate from DHAP and butanal. Insulation required in particular inactivation of the amn gene encoding the AMP nucleosidase, which otherwise led to glucose-independent DHAP production from adenosine phosphates. The work demonstrates that a sufficiently insulated in vitro multi-step enzymatic system can be readily assembled from central carbon metabolism pathways.

Intrinsic thermal sensing controls proteolysis of Yersinia virulence regulator RovA.

Pathogens, which alternate between environmental reservoirs and a mammalian host, frequently use thermal sensing devices to adjust virulence gene expression. Here, we identify the Yersinia virulence regulator RovA as a protein thermometer. Thermal shifts encountered upon host entry lead to a reversible conformational change of the autoactivator, which reduces its DNA-binding functions and renders it more susceptible for proteolysis. Cooperative binding of RovA to its target promoters is significantly reduced at 37 degrees C, indicating that temperature control of rovA transcription is primarily based on the autoregulatory loop. Thermally induced reduction of DNA-binding is accompanied by an enhanced degradation of RovA, primarily by the Lon protease. This process is also subject to growth phase control. Studies with modified/chimeric RovA proteins indicate that amino acid residues in the vicinity of the central DNA-binding domain are important for proteolytic susceptibility. Our results establish RovA as an intrinsic temperature-sensing protein in which thermally induced conformational changes interfere with DNA-binding capacity, and secondarily render RovA susceptible to proteolytic degradation.