Cell-Free Biosynthesis of Complex Molecules Using Designed and Tailored Permeabilized Cells

Complex low molecular weight compounds comprising polyketides, polypeptides, carbohydrates as well as combinations and derivatives thereof are presently regaining importance as weapons against various diseases like cancer, infections or chronic illnesses. Metabolic engineering activities have been directed to the biosynthesis of such compounds using natural producers but increasingly also model organisms as E. coli. Though the biological diversity is enormous and increasingly accessible using modern genetic engineering methods, there are some crucial limitations: (i) Usually the obtained final titers are low, (ii) chemically similar byproducts produced complicate down-stream processing, (iii) the creation of new compound variants is generally possible by engineering the core synthesis machines, usually megasynthases, but still very difficult and time consuming, and (iv) in vitrostudies of megasynthases are limited because of their inherent fragility.

We show that cell-free biosynthesis using permeabilized cells, in combination with computational tools for adequate pathway design, contributes significantly to solving problems sketched above and to develop organisms and processes for the production of complex low molecular weight compounds. Permeabilized cells still keep all their proteins in a state close to the original in vivo one. After removal of all original metabolites, a designed cocktail of substrates and cofactors is added to start biosynthesis.

First we developed computational tools to design metabolic networks supporting the cell-free biosynthesis. We start from a pan-organism metabolic network derived from databases as KEGG combined with thermodynamic data for reaction equilibria (eQuilibrator) and search for a biosynthesis path or network for a target molecule. A list of potential start metabolites and cofactors is used together with a set of constraints to identify pathway candidates using MILP. These candidates are then ranked using a set of criteria, e.g. length, thermodynamics or number of heterologous genes required. Promising candidates are finally evaluated by experts and implemented.

With some practical examples, where we applied engineered, permeabilized cells for production of uridinylated carbohydrates (UDP-glucose), polyketides (flaviolin and oxytetracycline) and non-ribosomal polypeptides (luminmides and new biosynthetic variants), we show that this methodology will significantly contribute to the production of complex low molecular weight compounds.