BackgroundAs more and more biological reaction data become available, the full exploration of the enzymatic potential for the synthesis of valuable products opens up exciting new opportunities but is becoming increasingly complex. The manual design of multi-step biosynthesis routes involving enzymes from different organisms is very challenging. To harness the full enzymatic potential, we developed a computational tool for the directed design of biosynthetic production pathways for multi-step catalysis with in vitro enzyme cascades, cell hydrolysates and permeabilized cells.ResultsWe present a method which encompasses the reconstruction of a genome-scale pan-organism metabolic network, path-finding and the ranking of the resulting pathway candidates for proposing suitable synthesis pathways. The network is based on reaction and reaction pair data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the thermodynamics calculator eQuilibrator. The pan-organism network is especially useful for finding the most suitable pathway to a target metabolite from a thermodynamic or economic standpoint. However, our method can be used with any network reconstruction, e.g. for a specific organism. We implemented a path-finding algorithm based on a mixed-integer linear program (MILP) which takes into account both topology and stoichiometry of the underlying network. Unlike other methods we do not specify a single starting metabolite, but our algorithm searches for pathways starting from arbitrary start metabolites to a target product of interest. Using a set of biochemical ranking criteria including pathway length, thermodynamics and other biological characteristics such as number of heterologous enzymes or cofactor requirement, it is possible to obtain well-designed meaningful pathway alternatives. In addition, a thermodynamic profile, the overall reactant balance and potential side reactions as well as an SBML file for visualization are generated for each pathway alternative.ConclusionWe present an in silico tool for the design of multi-enzyme biosynthetic production pathways starting from a pan-organism network. The method is highly customizable and each module can be adapted to the focus of the project at hand. This method is directly applicable for (i) in vitro enzyme cascades, (ii) cell hydrolysates and (iii) permeabilized cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-017-1773-y) contains supplementary material, which is available to authorized users.

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: Recent developments in the field of biocatalysis using permeabilized cells are reviewed here, with a special emphasis on the newly emerging area of multistep biocatalysis using permeabilized cells. New methods of metabolic engineering using in silico network design and new methods of genetic engineering provide the opportunity to design more complex biocatalysts for the synthesis of complex biomolecules. Methods for the permeabilization of cells are thoroughly reviewed. We provide an extended review of useful available databases and bioinformatics tools, particularly for setting up genome-scale reconstructed networks. Examples described include phosphorylated carbohydrates, sugar nucleotides, and polyketides.

We constructed and applied a recombinant, permeabilized Escherichia coli strain for the multistep synthesis of UDP-glucose. Sucrose phosphorylase (E.C. 2.4.1.7) of Leuconostoc mesenteroides was over expressed and the pgm gene encoding for phosphoglucomutase (E.C. 5.4.2.2) was deleted in E. coli to yield the E. coli JW 0675-1 SP strain. The cells were permeabilized with the detergent Triton X-100 at 0.05 % v/v. The synthesis of UDP-glucose with permeabilized cells was then optimized with regard to pH, cell density during the synthesis and growth phase during cell harvest, metal cofactor, other media components, and temperature. In one configuration sucrose, phosphate, UMP, and ATP were used as substrates. At pH 7.8, 27 mg/ml cell dry weight, cell harvest during the early stationary phase of growth and Mn(2+) as cofactor a yield of 37 % with respect to UMP was achieved at 33 °C. In a second step, ATP was regenerated by feeding glucose and using only catalytic amounts of ATP and NAD(+). A UDP-glucose yield of 60 % with respect to UMP was obtained using this setup. With the same setup but without addition of external ATP, the yield was 54%.

Cell‐free systems containing multiple enzymes are becoming an increasingly interesting tool for one‐pot syntheses of biochemical compounds. To extensively explore the enormous wealth of enzymes in the biological space, we present methods for assembling and curing data from databases to apply them for the prediction of pathway candidates for directed enzymatic synthesis. We use Kyoto Encyclopedia of Genes and Genomes to establish single organism models and a pan‐organism model that is combining the available data from all organisms listed there. We introduce a filtering scheme to remove data that are not suitable, for example, generic metabolites and general reactions. In addition, a valid stoichiometry of reactions is required for acceptance. The networks created are analyzed by graph theoretical methods to identify a set of metabolites that are potentially reachable from a defined set of starting metabolites. Thus, metabolites not connected to such starting metabolites cannot be produced unless new starting metabolites or reactions are introduced. The network models also comprise stoichiometric and thermodynamic data that allow the definition of constraints to identify potential pathways. The resulting data can be directly applied using existing or future pathway finding tools.