Finding Enzymes for Novel Synthetic Pathways Constructions to Generate High Value Added Chemicals

The low-hanging fruit for metabolic engineering targets have in many cases been picked and challenges in commercialization of low-value fuel production systems have resulted in a gradual shift in attention towards higher value products with expanding potential diversity. Synthetic biology allows us to create new metabolic pathways for industrial biocatalysis by transfer of existing “parts” available in nature, or to produce pathways towards molecules that naturally do not exist at all. A limiting factor for the creativity solution space is the availability of genes encoding suitable enzymes. Towards this objective, we have developed a protocol for structure-based prediction of the promiscuity of enzyme substrate specificity with the aim to narrow potential targets for secondary experimental trials in the construction of novel pathways. The protocol includes a phylogenetic search at the primary level, followed by structural model refinement (Ramachandran, ERRAT, GDT-HA, RMSD, Molprobity) and molecular docking using three complementary prediction algorithms and tools (Phyre2, Swiss Model, Autodock). The combined score provides an estimate of the predicted affinity (Kcal/mol) of the tested substrates. In vitro assays with purified recombinant novel enzymes are used to validate the molecular predictions. We will present case studies where we (i) have carried out in silico screening for substrate specificity of novel enzymes involved in synthetic pathways by molecular docking; (ii) evaluated these novel enzymes in vitro activities to validate the computational predictions.