A quantitative method for proteome reallocation using minimal regulatory interventions

Abstract

Engineering resource allocation in biological systems for synthetic biology applications is an ongoing challenge. Wild type organisms allocate abundant cellular resources for ensuring survival in changing environments, reducing the productivity of engineered functions. Here we present a novel approach for engineering the resource allocation of Escherichia coli by rationally modifying the transcriptional regulatory network of the bacterium. Our method (ReProMin) identifies the minimal set of genetic interventions that maximise the savings in cell resources that would normally be used to express non-essential genes. To this end we categorize Transcription Factors (TFs) according to the essentiality of the genes they regulate and we use available proteomic data to rank them based on its proteomic balance, defined as the net proteomic charge they release. Using a combinatorial approach, we design the removal of TFs that maximise the release of the proteomic charge and we validate the model predictions experimentally. Expression profiling of the resulting strain shows that our designed regulatory interventions are highly specific. We show that our resulting engineered strain containing only three mutations, theoretically releasing 0.5% of their proteome, has higher proteome budget and show increased production yield of a molecule of interest obtained from a recombinant metabolic pathway. This approach shows that combining whole-cell proteomic and regulatory data is an effective way of optimizing strains in a predictable way using conventional molecular methods.\n\nImportanceBiological regulatory mechanisms are complex and occur in hierarchical layers such as transcription, translation and post-translational mechanisms. We foresee the use of regulatory mechanism as a control layer that will aid in the design of cellular phenotypes. Our ability to engineer biological systems will be dependent on the understanding of how cells sense and respond to their environment at a system level. Few studies have tackled this issue and none of them in a rational way. By developing a workflow of engineering resource allocation based on our current knowledge of E. colis regulatory network, we pursue the objective of minimizing cell proteome using a minimal genetic intervention principle. We developed a method to rationally design a set of genetic interventions that reduce the hedging proteome allocation. Using available datasets of a model bacterium we were able to reallocate parts of the unused proteome in laboratory conditions to the production of an engineered task. We show that we are able to reduce the unused proteome (theoretically 0.5%) with only three regulatory mutations designed in a rational way, which results in strains with increased capabilities for recombinant expression of pathways of interest.\n\nHighlightsO_LIProteome reduction with minimal genetic intervention as design principle\nC_LIO_LIRegulatory and proteomic data integration to identify transcription factor activated proteome\nC_LIO_LIDeletion of the TF combination that reduces the greater proteomic load\nC_LIO_LIRegulatory interventions are highly specific\nC_LIO_LIDesigned strains show less burden, improved protein and violacein production\nC_LI